Research Projects -
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Development of periodontal tissue regeneration therapy for horizontal alveolar bone resorption with collagen-binding FGF-2
Grant number：19H03831 2019.04 - 2023.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
Takashiba Shogo
Grant amount：\17030000 （ Direct expense: \13100000 、 Indirect expense：\3930000 ）
Basic fibroblast growth factor (bFGF) has limited periodontal regenerative applications due to its low local fixation. We have shown that collagen-binding bFGF (CBFGF) in combination with collagen powder (CP) is effective in the treatment of horizontal alveolar bone defects in rats, and in this study we characterized CBFGF and evaluated the efficacy of CBFGF/CP in a canine model. CBFGF/CP increased the number of mesenchymal stem cell-like cells and cells expressing NANOG and PDGF receptor alpha on postoperative day 5, and promoted new bone and cementum formation in both vertical and horizontal bone defects. In conclusion, CBFGF/CP can greatly improve the limitations of conventional periodontal regenerative therapy.
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Grant number：22390397 2010 - 2012
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
TAKASHIBA Shogo, MAEDA Hiroshi, OHARA Naoya, NOZOE Mikio
Grant amount：\13000000 （ Direct expense: \10000000 、 Indirect expense：\3000000 ）
It is difficult to measure serum IgG antibody titers against periodontopathic bacteria stably and speedily. In this study, we havesucceeded to select antigens of Porphyromonas gingivalisfor effective serodiagnosis of periodontitis and synthesized 16 antigens. In addition, we confirmed the reaction against serum obtained from periodontal patients. Inthe future, we will identify antigens for effective serodiagnosis and establish the automatic diagnostic system.
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Mail Medicine using Fingertip Plasma for Screening and Monitoring Periodontitis
Grant number：18209061 2006 - 2008
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
TAKASHIBA Shogo, NAGATA Toshihiko, ABIKO Nobumitu, YAMAZAKI Kazuhisa, NAGASAWA Tosiyuki, HINO Takamune, YOSHIMURA Atsutoshi, SHIMAUCHI Hidetoshi, OGATA Yorimasa, NUMABE Yukihiro, NOGUCHI Toshihide, MURAKAMI Shinya, NARUISHI Koji
Grant amount：\48100000 （ Direct expense: \37000000 、 Indirect expense：\11100000 ）
我々は, 歯周病検査法としての歯周病原細菌に対する血漿IgG抗体価検査の有用性を検討した。P. gingivalis(Pg)などの4菌株を標的とした。また対象は慢性歯周炎患者549名とした。「BOP陽性率」および「4mm以上の歯周ポケットの割合」を各々3群に分類して各群の抗体価の有意差を調べた結果, Pgに対する血漿IgG抗体価は歯周病の悪化に相応して高値を示した。また「歯周基本治療後」群の抗体価(N=377)は, 「初診時」群の値と比較して4菌株すべての抗原において有意に減少した。すなわち, 本検査法は歯周病病態を評価し得る検査であると考える
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歯周病による腸内の鉄代謝異常が大腸癌の進行に与える影響の解明
Grant number：24K12912 2024.04 - 2027.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
平井 公人, 大原 利章, 高柴 正悟
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
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制御性T細胞の変化が関わるシェーグレン症候群特異的な新規非翻訳RNAの探索と解析
Grant number：22K09925 2022.04 - 2025.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
池田 淳史, 高柴 正悟, 伊藤 達男
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
シェーグレン症候群(SS)は唾液腺に生じる自己免疫疾患で、唾液分泌能の低下により齲蝕や摂食嚥下障害などが発症する。その結果、栄養摂取量の低下を介して健康寿命が短縮するため、社会的に大きな問題となっている。一般にSSを含めた自己免疫疾患の発症・進行には、制御性T細胞(Treg)の異常が関与している。既存のSSに関する研究報告の多くは、唾液腺中に存在するこのTregの数のみに着目し、DNAの塩基配列に依存しない遺伝子の調節機構であるエピジェネティクスの制御によるTregの機能的変化という観点では研究されていない。近年、疾患特異的なlong non-coding RNA(lncRNA)が、エピジェネティクスの制御などを介してTregに影響を与え、自己免疫疾患の発症や進行に関与していることが判明してきたが、SS特異的なlncRNAは発見されていない。
以上から、SS特異的に発現しているlncRNAによるエピジェネティックスの制御を介したTregの機能的変化が、SSの病態に関与しているのではないかという問いが生まれた。従って本研究の主目的を、SS特異的なlncRNA によってどのようにTregの機能が変化するか明らかにし、SSの発症・進行機序の一端を解明することに設定した。
まず、倫理委員会に本実験遂行にあたり、計画書を作成・提出し、承認を得た。そして、岡山大学病院リウマチ・膠原病内科の協力のもと、シェーグレン症候群患者のリクルートを行い、同意が得られた患者から採血を行い、岡山大学病院バイオバンクに資料を登録・保管した。
また、フローサイトメトリーを用いて、研究担当者自身の血液からTregの分離が的確に行えるか確認を行っている。 -
細胞外小胞の口腔トロピズムを基軸とする侵襲性歯周炎の病態解明と診断への応用展開
Grant number：21H03119 2021.04 - 2025.03
日本学術振興会 科学研究費助成事業 基盤研究(B)
山本 直史, 江口 傑徳, 宮地 孝明, 高柴 正悟, 江國 大輔, 井手口 英隆
Grant amount：\16900000 （ Direct expense: \13000000 、 Indirect expense：\3900000 ）
侵襲性歯周炎（Aggressive periodontitis：AgP）は全身的には健康な若年者に発症し，急速に進行する特殊な歯周炎であるが、その発症病態は不明なままである。本研究では，臓器特異的な作用（臓器トロピズム）が近年注目されている血中の細胞外小胞（EV）とAgPの病態関与の可能性を調べた。
今年度は、AgP患者6名と健常者3名の初診時血中EVから，AgPで高発現するmiRNAをRNAシーケンスにて調べ，マーカー候補となるそれらのmiRNA mimicをヒト歯肉線維芽細胞と歯周炎モデルマウスに遺伝子導入した。誘導された炎症性サイトカインの発現量をリアルタイムPCR法とELISA法にて測定し，歯槽骨吸収量をマイクロCTにて調べた。
健常者と比較して，AgP患者で発現量が2倍以上増加したmiRNAを500種類以上同定した。それらのうち5種のmiRNAとmiR-181b-5pを歯肉線維芽細胞に導入すると，IL-6とIL-1βの産生が増加した。とりわけ，miR-181b-5p を歯肉組織に導入すると歯槽骨吸収が進行した。
すなわち、AgP患者の血中EVには診断マーカー候補となるmiRNAが多く発現しており，miR-181b-5pはIL-6とIL-1β発現を伴う炎症を助長することによってAgPを重症化する可能性が示された。 -
細胞外小胞の口腔トロピズムを基軸とする侵襲性歯周炎の病態解明と診断への応用展開
Grant number：23K21486 2021.04 - 2025.03
日本学術振興会 科学研究費助成事業 基盤研究(B)
山本 直史, 江口 傑徳, 宮地 孝明, 高柴 正悟, 江國 大輔, 井手口 英隆
Grant amount：\16900000 （ Direct expense: \13000000 、 Indirect expense：\3900000 ）
侵襲性歯周炎（Aggressive periodontitis：AgP）は全身的には健康な若年者に発症し，急速に進行する特殊な歯周炎であるが、その発症病態は不明なままである。本研究では，臓器特異的な作用（臓器トロピズム）が近年注目されている血中の細胞外小胞（EV）とAgPの病態関与の可能性を調べた。
昨年度、AgP患者6名と健常者3名の初診時血中EVから，AgPで高発現するmiRNAをRNAシーケンスにて調べ、マーカー候補を同定した。今年度は、それらのmiRNA mimicを歯周炎モデルマウスに遺伝子導入し、炎症メカニズムを調べた。誘導された炎症性サイトカインの発現量をリアルタイムPCR法にて測定し，歯槽骨吸収量をマイクロCTにて調べた。
マーカ候補の5種のmiRNAとmiR-181b-5pをマウス歯肉組織に導入するとに導入すると，IL-6とIL-1βの産生が増加した。とりわけ，miR-181b-5p を歯肉組織に導入すると、M1マクロファージやTh1とTh17細胞が増加し，歯槽骨吸収が進行した。
すなわち、AgP患者の血中EVには診断マーカー候補となるmiRNAが多く発現しており，miR-181b-5pはIL-6とIL-1β発現を伴う炎症を助長することによってAgPを重症化する可能性が示された。
さらに、EVが内包する炎症性miRNAが歯周組織に到達するメカニズムに，血中EVの特異表面蛋白を介した臓器指向性が関与するとの仮説の下、EVのプロテオーム解析を行った。昨年度までに検証した様々なEV抽出方法と前処理法の結果を元にプロトコルを確立し、まずは健常者血中のEVのプロテオーム解析を行ったところ、多くのEVマーカーを含む2844蛋白質が同定・定量された。 -
プロトンポンプ阻害剤服用時に歯周病原細菌が腸内細菌叢へ及ぼす影響
Grant number：21K09893 2021.04 - 2024.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
平井 公人, 横田 憲治, 高柴 正悟
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
本研究の目的は胃酸分泌抑制剤であるプロトンポンプ阻害薬(PPI)の服用により胃酸の殺菌作用が低下した状態で，歯周病原細菌であるPorphyromonas. gingivalisもしくはその代謝産物が腸内細菌叢へ与える影響を調査することである。健康なマウスでは経口投与された細菌はほとんどが胃酸で殺菌されるが，PPI投与により胃酸の殺菌作用が低下した状態ではP.gingivalisは胃を生菌として通過し遠位腸管まで到達できるかどうかを検討した。
まずはPPIであるランソプラゾールのマウスへの経口投与が胃酸のpHをどの程度上昇させるかを検討するためにPPI投与後24時間後に安楽死させ切除した胃の内容物のpHを計測した。PPI投与群でも非投与群でもpHは2-3程度と差がなかった。これはマウスの餌の摂取制限ができないために胃内容物が多かったことが原因と考えられるため，今後はPPIの薬効の確認には血中ガストリン濃度の測定などで評価する必要がある。
歯周病感染モデルではマウスに1週間PPIの経口投与を行った後にP.gingivalisを2日間経口投与し，24時間後に盲腸の糞便を回収した。回収した糞便から約10mg採取し変法GAMブイヨン寒天培地上で嫌気培養し得られた菌体から採取したDNAと，盲腸糞便から直接採取したDNAを用いてP.gingivalisを特異的に認識するプライマーを用いてのDNA量をリアルタイムPCR法を用いて評価したとこと，PPIの有無に関わらず盲腸内で生菌としては確認されなかったが，盲腸内からはP.gingivalis遺伝子を確認することができた。また大腸組織の病理学的評価においてはPPI投与群で非投与群に比べてP.gingivalis経口投与によると思われる腸管粘膜の炎症性細胞浸潤や腸管上皮の傷害などの炎症所見が重症化する傾向にあった。 -
Development of novel protectant for oral mucosal disorder during cancer chemotherapy
Grant number：20333757 2020.08 - 2022.03
AMED 橋渡し研究戦略的推進プログラム・シーズB
大森 一弘, 高柴正悟, 入江 正郎, 堀綾花, 吉田道弘, 堀田勝幸, 本田成道, 小里達也, 山本裕也, 高木智久, 二村優次
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Pathogenesis of Rheumatoid Arthritis mediated by infection of periodontal pathogenic bacteria and citrullinated protein
Grant number：20K09954 2020.04 - 2023.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Hatanaka Kazu
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
To clarify the association between periodontitis and rheumatoid arthritis, we investigated infection of periodontal pathogenic bacteria involved in citrullination in patients with rheumatoid arthritis and similar diseases.
We found a significant association between elevated serum IgG antibody titers against periodontal bacteria P. gingivalis and anti-citrullinated peptide antibodies. Although there was no difference in disease activity before rheumatoid arthritis treatment and antibody titers against P. gingivalis and A. actinomycetemcomitans, patients with higher antibody titers had a poorer response to treatment after 3 months. No association was found for smoking, a risk factor for both diseases. It was suggested that infection with periodontal pathogenic bacteria may interfere with the therapeutic efficacy of rheumatoid arthritis. -
APPLICATION OF RvD2 AS A REGENERATIVE DIRECT PULP CAPPING MATERIAL
Grant number：20K09938 2020.04 - 2023.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
ARIAS MARTINEZ Zulema Rosalia
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
RvD2 applied directly to the pulpal surface, induced reparative dentin (RD) formation. The possible mechanism may be related to the RvD2-induced secretion of cytokines related to tissue regeneration such as VEGF and TGF-β in the pulp tissue thus promoting cell proliferation. RvD2 also decreased the TRAP1 pain receptor gene expression thus suppressing pain. These results suggest that RvD2 can be used for Vital pulp therapy (VPT) and may exert reparative actions by a mechanism different from that of existing VPT reagents, which do not induce inflammation. We conclude that RvD2 could be a material for the next generation VTP, comparable to pulp stem cell transplantation-based therapies.
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Identification and Functional Analysis of the Target Molecule of Terrein, a Small Molecule Compound for Application in the Super-Aging Society
Grant number：19K10108 2019.04 - 2023.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Omori Kazuhiro
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
In this study, we focused on terrein, a fungal secondary metabolite with anti-inflammatory properties, and investigated its effects on inflammatory bone-destroying pathologies both in vitro and in vivo, and explored the potential of terrein as a therapeutic agent for bone-destroying diseases. Our results showed that 1) terrein significantly suppressed the expression of NFATc1, a key factor for RANKL-induced osteoclast differentiation, and 2) the PKC pathway was involved in the suppression of NFATc1 expression. Furthermore, 3) TNF-alpha production was suppressed and alveolar bone resorption was inhibited in a mouse ligature-induced periodontitis model. These results provide a partial explanation for the anti-inflammatory and osteoclast differentiation inhibitory effects of terrein.
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Metabolic control of periodontal fibrotic diseases by fluorides: A basic study
Grant number：19K10150 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Osugi Ayaka
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
The purpose of this study was to verify whether fluorine ions, which have an anti-cariogenic effect, suppress the fibrosis of periodontal tissue.
Studies have shown that sodium fluoride (NaF) induces gene expression of the fibrotic molecule, cellular communication network factor (CCN) 2, and has no significant effect on the expression of the anti-fibrotic molecule CCN3. However, NaF suppresses the type I collagen gene, whose expression was increased by the induction of fibrosis by TGF-beta, as hypothesized.
Taken together, it was found that NaF confers a fibrosis-suppressing effect through an unknown mechanism, which is not mediated by CCN2. -
Development of a New Evaluation Method for Exercise Training Using Mitochondrial Activation of Peripheral Blood Mononuclear Cells
Grant number：18K19681 2018.06 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
Ogino Keiki
Grant amount：\6370000 （ Direct expense: \4900000 、 Indirect expense：\1470000 ）
The concept of mitohormesis, in which oxidative stress stimulation by exercise training leads to increased expression of antioxidant enzymes due to mitochondrial activation, leading to increased lifespan, was investigated in human peripheral blood mononuclear cells. In an intervention study with students, light exercise for 30 minutes daily for two weeks was found to increase the expression of SOD1mRNA and SOD2mRNA in peripheral blood mononuclear cells. Furthermore, in a cross-sectional study of approximately 392 individuals who underwent corporate health examinations at a health examination institution in Okayama Prefecture, SOD2mRNA and MtDNA were predominantly higher in humans with exercise habits. Multivariate analysis showed that exercise habit was associated with elevated SOD2mRNA, and that this association disappeared under the influence of smoking.
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Study on pathogenicity of Rothia mucilaginosa and development of the anti-infective therapy
Grant number：18K09613 2018.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Maeda Hiroshi
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
Distribution of Rothia species (Rothia mucilaginosa, Rothia aeria and Rothia dentocariosa), known to be opportunistic pathogens, in root canals was investigated. The Rothia spp. were frequently detected in root canals among Japanese population. The presence of Rothia spp. showed correlations with the inflammatory conditions at the apical lesions. The cell components of Rothia demonstrated high level of matrix metalloprotease activity as a potential virulence factor.
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Elucidation of pathology of periodontal disease, sarcopenia, and diabetes centered on inflammation-aging
Grant number：18K09598 2018.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Kobayashi Hiroya
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
Compared with mature mice (12 weeks old), aged mice (24 weeks old) tended to have more muscle tissue destruction by barium chloride. In the Porphyromonas gingivalis (P.g.) infected group, the number of necrotic cells in muscle tissue tended to be high.
Compared with the old model mouse with muscle injury, the periodontitis-old model mouse with muscle injury had a wider range of muscle tissue necrosis and tended to delay healing.
The expression level of IL-6 tended to be increased in the old model mouse with muscle injury, and the expression level tended to be higher in the old model mouse with periodontitis-muscle injury. -
Investigation of integrin peptide therapy regulating stem cell niche for periodontal regeneration
Grant number：18K09576 2018.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Yamamoto Tadashi
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
Extracellular matrix (ECM) and integrins-mediated microenvironments are important for the recruitment of tissue-resident stem cells. This study investigated the regenerative effects on periodontal tissue by integrin α3-blocking peptide (α325), previously identified as a migration factor of periodontal ligament cells and mesenchymal stem cells. In vivo study using rat horizontal bone defect model and mouse periodontitis model indicated that α325 induced alveolar bone regeneration, equal to or greater than FGF-2. Immunohistochemical analysis indicated that α325 induced mRNA expression of anti-inflammatory cytokines and stem cell marker genes and bone matrix proteins. This study concluded that α325 is a potent peptide-based drug, capable of anti-inflammatory effects and establishing local microenvironments for periodontal regeneration, defined by coordination between growth factors and ECM.
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Biological effect and preventive method for human serum albumin binding to transboundary air borne PM2.5.
Grant number：18H03039 2018.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
Ogino Keiki
Grant amount：\12870000 （ Direct expense: \9900000 、 Indirect expense：\2970000 ）
We discovered for the first time in the world that human albumin (hAlb) is bound to atmospheric fine particulate matter (PM2.5), and investigated its origin and biological effects. We found that hAlb, which is excreted from humans, binds to PM2.5, remains on the surface of the earth without being degraded, and may be dispersed into the atmosphere during winter when humidity is low. hAlb binds to PM2.5, enters cells through clathrin-dependent endocytosis, and induces oxidative stress. Furthermore, hAlb bound to PM2.5 reacted with O3 and NO2 in the air to form nitrated hAlb, which caused eosinophilia in bronchi via IL-5 and eotaxin-1.
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Grant number：16K11549 2016.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Omori Kazuhiro
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
In this study, we examined the possibility of the anti-inflammatory small molecule compound terrein as a therapeutic agent for inflammatory bone resorption diseases (especially endodontic or periodontal diseases). As a result of present study, (1) success of synthesized new terrein analogues, which have inhibitory effect of osteoclast differentiation, (2) one of the intracellular target molecules of terrain is JAK1, (3) intraperitoneal administration of terrein significantly inhibited alveolar bone resorption in the mouse ligature-induced periodontal disease model, and terrain suppressed the infiltration of inflammatory cells into the subepithelial region. The results suggest that terrein, a low molecular weight compound, may be applied as a treatment for endodontic or periodontal diseases.
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Functional analysis of HMGB1 as an inflammatory mediator in periodontitis
Grant number：16K20670 2016.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
YAMASHIRO KEISUKE, AOYAGI Hiroaki, IDEGUCHI Hidetaka, KOCHI Shinsuke, YAMAMOTO Tadashi, TAKASHIBA Shogo, WAKE Hidenori, NISHIBORI Masahiro
Grant amount：\3900000 （ Direct expense: \3000000 、 Indirect expense：\900000 ）
High mobility group box 1 (HMGB 1) is a DNA binding protein, but it plays as an inflammatory mediator when it is secreted extracellularly due to tissue damage or necrosis. The detailed mechanism of how HMGB1 affects the progress of periodontitis has not been elucidated. As a result of this study, it was revealed that HMGB1 was produced from gingival epithelial cells, macrophage-like cells by inflammatory stimulation. In addition, by administering anti-HMGB 1 antibody to periodontitis model mice, inflammation due to periodontitis is suppressed. As a result, migration of neutrophils, production of IL-1βwere suppressed and the bone resorption by periodontitis was suppressed.
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Grant number：15K11404 2015.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
MAEDA Hiroshi
Grant amount：\4940000 （ Direct expense: \3800000 、 Indirect expense：\1140000 ）
In this research project, we aimed to inhibit the growth of selected bacterial species in oral microflora by using antisense PNA (peptide nucleic acids). By targeting the HSP and AcpP genes of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, the antisense PNAs were designed and synthesized with carrier paptide (KFFKFFKFFK). For P. gingivalis, both antisense PNAs effectively inhibited the growth. Especially, anti-HSP PNA completely inhibited the growth for 5 hours. Comparing to P. gingivalis, the inhibit effect was weak for A. actinomycetemcomitans. Anti-HSP PNA slightly inhibit the growth, while anti-AcpP PNA did not show the effect. The cause of the low effect may be due to the small uptake of the PNA in A. actinomycetemcomitans. Antisense PNA may have the potential to reduce the population of P. gingivalis in oral microflora. New designs for carrier peptide and the target genes will be required for A. actinomycetemcomitans.
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Grant number：26462881 2014.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
SOGA Yoshihiko, MAEDA Yoshinobu, MURO Misato, HIGUCHI Tomoko, OKUI Akemi, KATAOKA Kota, EKUNI Daisuke, TANIMOTO Mitsune, IIDA Seiji, MORITA Manabu, MATSUDA Yuri, KAWAMURA Yumeno, Yasuoka Rika, TAKEDA Yuriko, MISHIMA Misuzu, KATAYAMA Tomoko, ONO Yoshiko, TAKAHASHI Kanayo, KONDO Eisei, FUJII Nobuharu, TAO Ayaka, SATO Takamaro, FUJII Yurie, MIYAOKA Mana, MUKAI Mariko, KODAMA Yuka, TAKEMORO Nana, TAKASHIBA Shogo, MORI Takehiko, HOSOKAWA Ryoichi, NASU Junichiro, MATSUBARA Minoru, MIURA Ko, KANZAKI Hiromitsu, OKADA Hiroyuki, YAMAMOTO Kazuhide, SUGIURA Yuko
Grant amount：\5070000 （ Direct expense: \3900000 、 Indirect expense：\1170000 ）
1) Febrile neutropenia caused by odontogenic infection was identified. ericoronitis was suspected as the origin of febrile neutropenia, and needs of further study was indicated.
2) The oral mucosal microbiota in cases treated with strong antibiotics, such as glycopeptides, differs from normal. As ulcerative oral mucositis was observed only in unusual mucosal microbiota, it may be associated with progression of oral mucositis. These unexpected bacteria may be involved in the pathophysiology of oral mucositis, and in general infection, including febrile neutropenia, via oral mucositis.
3) The same bacteria isolates were identified in the blood and oral mucosa in a patient with infective endocarditis. This observation suggests that there is a route between the focus of infection with infective endocarditis and the oral cavity. -
Induction of Migration of Periodontal Ligament Cells by Selective Regulation of Integrin Expression
Grant number：26463134 2014.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Yamamoto Tadashi
Grant amount：\4810000 （ Direct expense: \3700000 、 Indirect expense：\1110000 ）
Periodontal ligament cells (PDLCs) are multipotent cells that can differentiate into osteogenic cells. It is necessary to induce migration of PDLCs for regeneration and homeostasis of periodontal tissue. Cell migration is regulated by local microenvironment, defined by coordination between soluble factors and extracellular matrix (ECM). Integrin-mediated cell adhesion to ECM provides essential signals for cell migration. To determine adhesion molecules responsible for migration of PDLCs, we examined expression profiles of integrin and ECM, and the integrin isoform-specific regulation of migration of PDLCs. The array analysis indicated that mRNA of integrin α2, α3, α4, and α5 were increased in migrating PDLCs. Anti-integrin α3 antibody and blocking peptides significantly increased migration of PDLCs, conversely anti-integrin α5 antibody decreased. Therefore, specific inhibition of integrin α3 may be useful to induce migration of PDLCs during regeneration of periodontal tissue.
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Grant number：25253104 2013.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
YOSHIE Hiromasa
Grant amount：\44980000 （ Direct expense: \34600000 、 Indirect expense：\10380000 ）
The aim of this study was to identify the shared risk cytokine gene for periodontitis, diabetes mellitus (DM), and rheumatoid arthritis (RA). A total of 17 candidate single nucleotide polymorphisms were assessed in 185 patients with RA and chronic periodontitis (CP), 149 patients with type 2 DM and CP, 251 patients with CP, and 130 systemically and periodontally healthy controls from a cohort of Japanese adults. The results indicated that the potassium voltage-gated channel KQT-like subfamily, member 1 (KCNQ1) rs2237892 and the peptidylarginine deiminase type 4 (PADI4)_104 rs1748033 polymorphisms were significantly associated with the comorbidity of RA and CP. Additionally, a trend toward increase was shown in the serum levels of anti-cyclic citrullinated peptide immunoglobulin G in the patients with RA and CP as compared to those with RA only. These results suggest that the KCNQ1 and possibly the PADI4_104 may constitute a shared risk gene for RA and CP in Japanese adults.
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唾液腺体性幹細胞とiPS細胞を用いた唾液腺機能再生に関する研究
Grant number：25463217 2013.04 - 2014.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
峯柴 淳二, 大森 一弘, 山本 直史, 高柴 正悟
Grant amount：\4940000 （ Direct expense: \3800000 、 Indirect expense：\1140000 ）
【研究の目的】唾液は，口腔感染制御を含めて口腔内環境を保つ重要な働きを持つ。しかし唾液を分泌する唾液腺は，自己再生能が低く，障害後の機能回復は難しい。我々は，CD49F+細胞がin vitroではINHIBIN βA，INHIBIN βB，FOLLISTATINを発現することを報告している。INHIBINのβ鎖はホモ二量体を構成し，ACTIVIN分子と成る。一方FOLLISTATINは，ACTIVINに特異的に結合し，その受容体への結合を阻害する。本研究は，マウス顎下腺の主排泄導管を結紮後に解除すると顎下腺が再生することを利用し，in vivoにおいて唾液腺組織再生中のCD49F，INHIBIN βA，INHIBIN βB，そしてFOLLISTATINの発現局在の解明を目的とした。
【研究実施計画および結果】
マウス顎下腺の片側の排泄導管を血管結紮用クリップで結紮，他方は対照とし，6日後に結紮を解除した。結紮解除1，2，4，8，16日後の顎下腺を摘出し，パラフィン包埋切片作製の後，INHIBIN βA，INHIBIN βB，CD49FそしてFOLLISTATINの局在を免疫組織染色法で検討した。その結果，結紮解除後のどの日数でもINHIBIN βAは染色されず，INHIBIN βBとCD49fは染色された。また，結紮解除後8日目にはFOLLISTATINが染色された。さらに連続切片上で，CD49F，INHIBIN βB，そしてFOLLISTATINが同部位で染色された。以上から，結紮解除後8日目以降の唾液腺組織再生に，CD49F+細胞でのactivin-follistatin相互作用の関与を想定できる。
以上から本研究を行った結果，マウス顎下腺主排泄導管結紮解除後8日目の導管上皮細胞で，CD49F，INHIBIN βB，FOLLISTATINが発現していることが解明された。 -
Application of antisense PNA as antibiotics against periodontal pathogens
Grant number：24659925 2012.04 - 2015.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
MAEDA Hiroshi, TAKASHIBA Shogo, KOKEGUCHI Susumu, KITAMATSU Mizuki, YAMASHIRO Keisuke, MINESHIBA Fumi, ISOSHIMA Daichi
Grant amount：\3510000 （ Direct expense: \2700000 、 Indirect expense：\810000 ）
The final goal of this research project is to design peptide nucleic acids (PNA) with a carrier peptide and apply them as species-specific antisense drugs against periodontal pathogens. Prior to the application of antisense PNA, effective carrier peptides were selected. Total of 64 peptides (10 amino acids) were designed by referring to previous reports and were synthesized. Fluorescent label (tmp-red) was added to each peptide, and bacterial uptake of the peptides was examined by measuring the strength of the fluorescence. The synthesized peptides were incubated with periodontal pathogen Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Escherichia coli, and the fluorescence inside the bacterial cells was measured. As a results, a peptide with the sequence of Tmr-KFFKFFKFFK-NH2 demonstrated the most effective uptake of P. gingivalis. This peptide will be an effective carrier for antisense PNA against P. gingivalis.
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Grant number：24659924 2012.04 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
TAKASHIBA Shogo
Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）
Oral rehabilitation with oral implant has been widely performed with less consideration for infection of oral bacteria. Thus, it is reasonable to assume that latent oral infection such as periodontitis affects the prognosis of oral rehabilitation with oral implant.
This research project drew up a clinical research plan in order to clarify this clinical question. Both a research plan named as "An evaluation of oral bacterial infection before and after oral rehabilitation with oral implant using plasma IgG titer test against periodontopathic bacteria" and a comprehensive evaluation method for the change of oral microflora were proposed. -
Grant number：23593058 2011 - 2013
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
HATANAKA KAZU, TAKASHIBA Shogo, YAMAMOTO Todashi, YAMASHIRO Keisuke
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
The phenomenon of osteonecrosis of the jaw (ONJ) has been reported in the cancer patient who has received medication of the bisphosphonate (BP) to bone metastases. In Okayama University Hospital, the oral hygienist has been stationed in the Center for Clinical Oncology, and investigated the intraoral trouble. As a result, the Center use patients and the number of interviews by oral hygienist are increasing year by year, and were able to find six ONJ newly in three years. Those patients are followed up in the Department of Periodontics and Oral Surgery. Moreover, many of other chemotherapy patients had the symptom of stomatitis.
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Cell cycle atlas of periodontium
Grant number：23659977 2011 - 2013
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
YAMAMOTO Tadashi, TAKASHIBA Shogo, HATANAKA Kazu, YAMASHIRO Keisuke, YAMAGUCHI Tomoko
Grant amount：\3510000 （ Direct expense: \2700000 、 Indirect expense：\810000 ）
Fucci transgenic mice constitutively expressing cell-cycle probes are important tools to analyze the spatiotemporal transition of the cell-cycles in periodontium, especially, are useful to visualize the cell-cycles of undifferentiated mesenchymal cells and gingival epithelial cells. The molecular imaging employing Fucci technology demonstrated that a series of active enzymes produced by neutrophil during periodontal inflammation would induce G1-arrest of the cell-cycle of periodontal cells.
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2011 - 2012
産学が連携した研究開発成果の展開 研究成果展開事業 研究成果最適展開支援プログラム(A-STEP) 探索タイプ
高柴 正悟
本研究では、岡山県の特産品で安価に原料供給を受けることが出来るマッシュルームの石づき(廃棄部分)を原材料として、細菌バイオフィルムを抑制するレクチンの応用に関して、1安価で安定した大量抽出技術を確立し、2抽出物がStreptococcus mutans(S. mutans)以外のう蝕原因菌等の口腔細菌のバイオフィルム形成に対する抑制効果を検討して、本製法で得たレクチンを用いた口腔ケア剤がコスト的にも有効性においても実用的であることを検証する。本年度の研究では、マッシュルーム石づきからレクチン含有エキス製造法の確立を目標として開発研究を進めてきた。評価方法としては、赤血球凝集測定によって抽出物のレクチン活性を評価した。現段階では目標値にほぼ達する行程を確立することができており、今後バイオフィルム抑制効果および他菌種への検討へと移行する。
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How periodontitis can affect prostate cancer metastasis?
Grant number：22592312 2010 - 2012
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
TANIMOTO Ichiro, WATANABE Masami, NARUISHI Koji, MINESHIBA Junji, OMORI Kazuhiro, TAKASHIBA Syogo
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
In the present study, we demonstrated the opposite association between localized and systemic inflammatory responses. Our finding suggests that appropriate infectious exposure might be needed to maintain our life. Taken together, a severe sepsis-like condition elicited by bacteremia, is likely responsible for the mortality associated with P. gingivalisinfection, but immune system would regulate the unwanted systemic responses. These systemic responses may alsoimpact metastatic cancer.
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Grant number：22890119 2010 - 2011
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up
YAMASHIRO Keisuke, YAMAMOTO Tadashi, TAKASHIBA Shogo
Grant amount：\2977000 （ Direct expense: \2290000 、 Indirect expense：\687000 ）
Periodontal disease, chronic inflammation disease, is caused by infection of periodontal pathogen. It is thought that about 80% of Japanese are affected and it is a major cause of tooth loss. It is still unknown that the difference of progression of periodontal disease for each patient. It is thought that the attachment between gingival epithelial cells and tooth is a physical barrier from infection, and this attachment is important for prevention of periodontal disease. In this study, we hypothesis that growth factors secretes from gingival epithelial cells regulate the attachment between gingival epithelial cells and tooth, and we investigate the mechanism about the regulation.
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Chronic infection and immune response in COPD patients
Grant number：21590964 2009 - 2011
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
MURO Shigeo, ITO Isao, NARUISHI Koji, SATO Susumu, TAKASHIBA Shogo, MISHIMA Michiaki
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
Our prospective observational study including 93 COPD patients showed that the numbers of exacerbations and the rate of individuals with frequent exacerbations(at least two per year) were significantly lower in patients with higher IgG titer than those with normal IgG titer. Thus in contrast to our hypothesis, normal-IgG titer for periodontitis-related antibody can be an independent predictor of frequent exacerbations suggesting that humoral immune response associates with COPD exacerbations. We also found that COPD exacerbation accelerated the emphysema progression in spite of current additional therapy during exacerbations.
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Grant number：21592624 2009 - 2011
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
MAEDA Hiroshi, TAKASHIBA Shogo, KOKEGUCHI Susumu, TANIMOTO Ichiro
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
Periodontitis is known to be an infectious disease caused by oral bacteria. Besides bacteria, it has recently been reported that methanogenic Archaea is also involved in the pathogenesis of periodontitis. Archaea possess group II chaperonin that is homologous to human chaperonin CCT. Due to the sequence similarity, archaeal chaperonin has the potential to be a cross-reactive antigen of human CCT. We examined the reactivity of sera from patients with periodontitis or autoimmune diseases to the chaperonins. Antibody to the archaeal chaperonin and human CCT was detected in some patients. The results demonstrated the possible induction of autoimmune reaction targeting the group II chaperonin. Cross reaction between the archaeal and human chaperonin may be a trigger for autoimmune reaction.
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Grant number：20592429 2008 - 2010
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
YAMAMOTO Tadashi, TAKASHIBA Shogo, MINESHIBA Junji, YAMASHIRO Keisuke, NARUISHI Koji, SHIOMI Nobuyuki, SONOYAMA Wataru
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
In periodontal ligament cells (PDL cells), cytoskeletal signaling regulated by Rho-ROCK is critical in the differentiation process, and the expression of osteogenic genes such as BMP-4, Wnt3a, and Wnt5a. Meanwhile over-expression of RhoA or ROCK showed significant low cell proliferation, suggesting that PDL cells differentiation is coordinately regulated by Rho-ROCK induced cytoskeletal molecules, as well as soluble growth factors and extracellular matrix.
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Possible Mechanisms of Progression of Periodontits by MMP-3 in Diabetic Patients
Grant number：20592428 2008 - 2010
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
NARUISHI Koji, HATANAKA Kazu, TAKASHIBA Syougo, MINESHIBA Junji, OMORI Kazuhiro
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
Diabetic patients are susceptible to severe inflammatory periodontitis. In the present study, we found that high glucose increased MMP-3, but not sIL-6R production in gingival fibroblasts. In addition, sIL-6R production was suppressed by MMP-3 inhibitor in THP-1 macrophages. These results suggest that MMP-3 has a key role in developing severe periodontitis in diabetic patients. Furthermore, it has been considered that IL-6 signals are very important factor surrounding gingival fibroblasts/macrophage interaction in severe periodontitis seen in diabetic patients.
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Healing mechanism of rat experimental periapical lesions targeting laminin γ2 expression
Grant number：20592226 2008 - 2010
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
HATANAKA Kazu, YAMAMOTO Tadashi, TAKASHIBA Shougo, SHIMOE Masayuki, YAMAGUCHI Tomoko, NARUISHI Koji, KAKO Aya
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
We have previously reported that cellular matrix laminin and inflammatory cytokine IL-1αgenes increased during periapical healing phase in a rat periapical periodontitis. In this study, we investigated the effect of these molecules to osteoblast that play a central role in bone formation. Our findings the enhancement of integrin α3 expression by IL-1α and the attachment of osteoblasts to laminin after stimulation with IL-1α suggest an important mechanism for adherence of osteoblasts in periapical healing.
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付着歯肉の分化に関連した特異的遺伝子・蛋白の同定とその機能解析
Grant number：19659507 2007 - 2008
日本学術振興会 科学研究費助成事業 萌芽研究
窪木 拓男, 高柴 正悟, 園山 亘, 滝川 正春
Grant amount：\3200000 （ Direct expense: \3200000 ）
1.付着歯肉組織と遊離歯肉組織の遺伝子発現解析
前年度のマウスならびにラット歯肉組織の組織学的検討をもとにして,成体マウスの口腔内から実態顕微鏡下で付着歯肉と遊離歯肉をそれぞれ採取した、この組織からmRNAを抽出し,cDNAマイクロアレイを行い,両者の遺伝子発現を比較・検討した.
その結果,付着歯肉で発現量の高い遺伝子として,mmp12, integrin(alpha6), laminin(beta3), HIF-1a, VEGF, tenomodulin, collagen(typeV, alpha2), integrin(beta4)などが抽出された.一方,遊離歯肉で発現量の高い遺伝子としてIGFBP2, RABL3(member of RAS oncogene family-like3), RASA3(RAS p21 protein activator3), elastinなどが抽出された.既知の発現パターンと機能から考察するといくつかの遺伝子はたいへん興味深い研究対象と考えられた.
2,ヒト歯肉上皮細胞の培養
倫理委員会の許可を得て,抜歯時に得られたヒト歯肉サンプルから歯肉上皮細胞を分離し,同時に得た歯原性上皮細胞とその差異を比較,検討した.
その結果,歯肉上皮細胞は培養条件下では寿命が短く,cumulative population doubling(cPD)は平均8であった。一方,歯原性上皮細胞は平均16のcPDを示した.また,両者ともに上皮細胞のマーカーであるサイトケラチン14とE-cadherinを遺伝子レベルで発現していたが,amelogeninの発現は歯肉上皮細胞では認めなかった.すなわち,歯肉上皮細胞は歯原性上皮細胞と比較して,明らかに異なるフェノタイプを有していることを明らかにした. -
Molecular cloning and functional analysis of non-coding RNA expressed in periodontal bacteria
Grant number：19592387 2007 - 2008
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
MAEDA Hiroshi, TAKASHIBA Syogo, ARAI Hideo, TANIMOTO Ichiro, SOGA Yoshihiko
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
歯周病の原因となる細菌(歯周病細菌)における、非翻訳RNAの役割を解析することを目的として、以下の研究成果を得た。(1) 歯周病細菌Aggregatibacter actinomycetetemcomitansに大腸菌と類似した非翻訳RNAが発現していることを示した。(2) A. actinomycetemcomitansからRNAシャペロンを同定し、クローニングした。(3) RNAシャペロンを介した非翻訳RNAによる遺伝子発現調節機構がA. actinomycetemcomitansに存在する可能性を示した。(4)歯周病の病態には細菌だけでなく古細菌種が関与しており、その解析の必要性を示した。
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New bactericide delivery system for oral care
Grant number：19592201 2007 - 2008
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
TANIMOTO Ichiro, SHIOMI Nobuyuki, NARUISHI Koji, TAKASHIBA Syogo, MAEDA Hiroshi
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
口腔内の二大感染症であるう蝕と歯周病を効果的に予防するため, 歯面にとどまる殺菌剤と新規担体の組み合わせを開発した。塩化セチルピリジニウム(CPC)とリン酸化プルランの混合物は, リン酸化アパタイト表面に吸着し, う蝕原性細菌・歯周病原細菌に対して抗菌作用を発揮することが明らかになった。新規物質であるリン酸化プルランの安全性をラットの肝臓で確かめ, 為害性が少ない物質であることを確認した。
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The wound healing mechanisms and regenerative therapy in dental pulp and periapical tissues
Grant number：18209057 2006 - 2008
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
YOSHIMINE Yoshito, KITAMURA Chiaki, SHIBA Hideki, KAWASHIMA Nobuyuki, DOKUDA Masayuki, TAKASHIBA Syogo, MAEDA Katumasa, YOKOSE Satoshi, SYOJI Shigeru, SAITO Takashi, KUNIMATSU Kazushi
Grant amount：\45110000 （ Direct expense: \34700000 、 Indirect expense：\10410000 ）
歯の神経(歯髄)や根の先の周りの骨(根尖部歯周組織)に異常が生じる疾患において、これらの傷害が治癒するメカニズムを詳細に調べることで、従来とは異なる新しい治療法の確立に向けた包括的な研究を試みた。その結果、歯髄・象牙質・骨組織の再生への足がかりとなるデータを多く得ることができた。今後更に研究を発展させることで、臨床応用の可能な治療法の開発へと繋がるものと期待される。
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マイクロバブルを用いた口腔嫌気性菌除去方法の検討
Grant number：18659623 2006 - 2007
日本学術振興会 科学研究費助成事業 萌芽研究
高柴 正悟, 谷本 一郎, 前田 博史
Grant amount：\2000000 （ Direct expense: \2000000 ）
平成19年度の研究課題に関する研究実績は以下のとおりである。
1.マイクロバブル濃度の測定とバブル径の測定
平成18年度の研究では,市販のマイクロバブル水にはほとんど抗菌性のないことが明らかとなった。原因としては,マイクロバブル濃度が低いこと,そしてバブルの径が大きくマイクロバブルとしての作用が発現していないことが考えられた。このため,高速ビデオ撮影装置を応用して,バブル発生状況を調べた。その結果,市販の装置では直径がナノメーターあるいはマイクロメーターレベルのバブルはほとんど発生していないことが明らかとなった。そこで,本学工学部(柳瀬眞一郎)に依頼し,工学部で開発されたマイクロバブル発生装置を用いて,以下の抗菌試験と,ヒト細胞への影響について調べた。
2.歯周病細菌に対する抗菌試験
歯周病細菌Porphyromonas gingivalisを対数増殖期まで培養し,培養液5ccに対して1ccのマイクロバブル水を添加し,その後の菌増殖を培養液の吸光度で評価した。その結果,菌増殖はコントロール(蒸留水)とマクロバブル水の間で差がなく,マイクロバブル水にはほとんど抗菌性のないことが示された。
3.ヒト細胞への影響
ヒト上皮系細胞(HeLa)と単球系細胞(THP-1)の培養液中にマイクロバブル水を添加して,2時間細胞を培養した。その後,細胞を回収して,マイクロバブルが細胞のサイトカインならびに増殖因子発現に与える影響をプロテインアレイ法で解析した。その結果,マイクロバブルを添加した細胞のサイトカインプロファイルと増殖因子プロファイルはコントロールと比較して変化がなく,マイクロバブルによる影響はないことが示唆された。
これらの結果はマイクロバブルによる短時間の刺激では、細菌や細胞へ与える影響がほとんどないことを示すものである。今後は洗浄効果(プラーク除去)を中心にマイクロバブルの口腔内応用を考えていく必要がある。 -
Establishment of Information Basis for Tooth Regeneration
Grant number：17209062 2005 - 2008
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
KUBOKI Takuo, UEDA Minoru, KANYAMA Manabu, TAKASHIBA Syogo, TSUJI Takashi, TAKIGAWA Masaharu, ASAHAR Hiroshi, TUCHIMOTO Youhei, SONOYAMA Wataru, TAGAWA Youichi
Grant amount：\48880000 （ Direct expense: \37600000 、 Indirect expense：\11280000 ）
マウスの歯の発生時に認められる遺伝子を検索し、従来報告のなかった28個の遺伝子を同定した。エナメル質形成細胞の成熟は、周囲に存在する細胞が制御していることを証明した。高脂血症治療薬(スタチン)は、象牙質の形成を促進し、歯科治療薬として応用しうることを示した。顎骨に存在する細胞は、手足の骨の細胞とは異なる性質を有していること、また、顎骨の再生促進に成長因子(結合組織成長因子、塩基性線維芽細胞増殖因子)が応用可能であることを確認した。
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New Approach to the Study on Oral Biofilm Infectious Diseases by Metagenomic Analysis
Grant number：17390502 2005 - 2007
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
FUKUI Kazuhiro, KOKEGUCHI Susumu, TANIMOTO Ichiro, TAKASHIBA Shogo, MAEDA Hiroshi, KARIYAMA Reiko
Grant amount：\16260000 （ Direct expense: \15300000 、 Indirect expense：\960000 ）
Oral microflora has been investigated so far with cultivation-based techniques. Over 700 species of bacteria have been previously estimated in oral cavity but only about 50% of them have been cultivated. Any understanding of the oral environment requires knowledge of the entire bacterial community. The uncultivated and as-yet-uncharacterized bacterial species may participate in the etiology of oral diseases. To resolve this problem, we devised an approach by the metagenomic analysis based on the bacterial 16S ribosomal RNA gene (16S rDNA) .
In this study, we determined the profiles, composition and changes of various oral microflora using culture-independent molecular methods, i.e., clone library method, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) method and terminal restriction fragment length polymorphism analysis (T-RFLP) method. We also established the high-resolution, real-time and three-dimensional imaging system on the biofilm structure and development in the modified capillary flow-cell using confocal laser scanning microscopy with fluorescence visualization supporting by Professor Philip S. Stewart (Center for Biofilm Engineering at Montana State University).
We analyzed the microbial profiles and composition of tonsilloliths, which are potential cause of oral malodor by 16S rDNAs based clone library method. The isolated partial 16S rDNA sequences (approximately 600bp) were analyzed. Anaerobic bacteria detected in tonsilloliths, belonged to the genera Fusobacterium, Eubacterium, Megasphaera, Porphyromonas, Prevotella, Selenomonas and Tannerella, which appear to be associated with production of volatile sulfur compounds. We further examined the effectiveness of professional toothbrushing on microflora changes in subgingival plaques by PCR-DGGE analysis. PCR-DGGE analysis revealed that professional toothbrushing resulted in a decrease in the number of periodontal pathogens and the dramatic change of microflora in subgingival plaques.
We also revealed that Archaea was frequently isolated from deep periodontal pockets in Japanese patients with periodontitis and pathogenic and opportunistic bacterial species were frequently detected in the patients receiving bone marrow transplantation after chemotherapy. -
The effect on dentin-pulp complex by FIP-2 isolated from rat wounded pulp
Grant number：17591991 2005 - 2006
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
ARAI Hideo, TAKASHIBA Shogo, NISHIMURA Fusanori, NARUISHI Koji, TANIMOTO Ichiro, MAEDA Hiroshi
Grant amount：\3500000 （ Direct expense: \3500000 ）
Pulpal wound healing followed by cavity preparation may involve reactionary or reparative dentinogenesis in relation to the cavity position; however, little is known about the molecular responses. We aimed to isolate and analyze genes induced or suppressed in the wounded pulp to identify molecular processes involved in the pulp responses to injury. Twenty-three cDNAs were isolated by cDNA subtraction between healthy and wounded pulp of rats. By library screening, we identified rat 14.7K-interacting protein (rFIP)-2A and B genes homologous to human FIP-2, being involved in regulating membrane trafficking and cellular morphogenesis. RT-PCR analysis showed induction for only rFIP-2B in the wounded pulp. In situ hybridization analysis revealed unique expression of rFIP-2s in adult and embryonic tissues of rats. Transcription of rFIP-2A and B was regulated by alternative use of promoters at rFIP-2 locus. When the rFIP-2A or B-pAcGFP1-Golgi construct was transfected into normal rat kidney (NRK-52E) cells, rFIP-2B was localized in Golgi of whereas rFIP-2A, which is a truncated protein lacking the N-terminal 250 amino acids of rFIP-2B, existed ubiquitously in the cytoplasm. In rat pulp fibroblasts (RPC-C2A) cells, rFIP-2B was significantly induced by tumor necrosis factor (TNF)-α, and the induction was dependent on c-jun N-terminal kinase (JNK) pathway. rFIP-2B was localized in the cytoplasm, and translocated into the nucleus by cell death stimuli. The results suggest that rFIP-2 expression is regulated by the alternative promoter site, and rFIP-2B is a crucial molecule mediated by TNF-a, may be involved in cell death pathway during pulp inflammation.
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カテプシン-Lプロモーターの薬剤応答配列を標的とした歯肉増殖症の治療法開発
Grant number：17659657 2005 - 2006
日本学術振興会 科学研究費助成事業 萌芽研究
西村 英紀, 高柴 正悟, 畑中 加珠, 小柳津 功介
Grant amount：\3300000 （ Direct expense: \3300000 ）
薬物性宙肉増殖症の病巣局所には細胞外基質が多量に蓄積し、病変歯肉組織は高度に線維化している。申請者らは過去に、本疾患を惹起する3種類の薬剤すべてが歯肉線維芽細胞においてライソゾーム酵素カテプシンーLの活性を遺伝子の転写レベルで抑制することを報告した(Nishimura F et al.,Am J Pathol,2002)。カテプシンーLは炎症性サイトカインーIL-6やMCP-1によってその遺伝子発現が調節されていると言われている。そこで、歯肉線維芽細胞においてIL-6やMCP-1を発現'させるモデルとしてHLAクラスII抗原を介した刺激を用い、これらサイトカインの調節機構を明らかにした。歯肉線維芽細胞上のHLAクラスII抗原はfocal adhesion kinase(FAK)と会合しており、HLAクラズII抗原を介した刺激でFAKがリン酸化を受け、IL-6やMCP-1が産生されることを明らかにした。FAKのリン酸化を特異的に阻害するといわれるルテオリンを作用させると、FAKのリン酸化が濃度依存性に抑制されるとともに、 HLAクラスII抗原を介した刺激によって誘導されるIL-6やMCP-1の産生量が低下した。これらのことからルテオリンによる歯肉線維芽細胞中のFAKリン酸化阻害作用が、梅肉増殖症惹起薬剤の作用と類似の効果を及ぼすことで結果的にカテプシンーL活性が抑制され、細胞外基質が蓄積する可能性が示唆された。これら3種類の薬剤はいずれも細胞内へのカルシウムイオンの流入を阻害することが知られている。今後、ルテオリンによるFAKリン酸化阻害作用がカルシウムイオンの流入阻害を介したものかどかを確認する必要がある。
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歯周病細菌に対する血清抗体価測定法の標準化に関する調査研究
Grant number：17639021 2005
日本学術振興会 科学研究費助成事業 基盤研究(C)
高柴 正悟, 永田 俊彦, 安孫子 宣光, 山崎 和久, 長澤 敏行, 日野 孝宗
Grant amount：\3300000 （ Direct expense: \3300000 ）
採血および検査方法の改善,標準化したデータベースの作成,臨床的に有効である根拠の探求を,基礎科学的問題,臨床的および社会的問題,産学官連携上の問題の観点から,6人の研究者が担当して,これらを組み合わせて9つの観点から検討した。
大規模臨床研究を実施する体制を,日本歯周病学会でのWGを中心に産学官の連携が成り立つように作成した。なお,米国において口腔内細菌と歯周病の病状を虚血性心疾患の罹患と重症度の関連をみる大規模臨床研究を行っているノースカロライナ大学チャペルヒル校の状況を,研究体制の樹立のモデルとして用い,さらに,基礎的な研究を臨床研究に発展させて検査と治療法の開発を行っている国立衛生研究所顎顔面歯科部門(NIDCR)における研究の展開の仕方をモデルとして用いた。そのために,これらの施設において研修を受けた日本人研究者から資料収集を行い,研究遂行上の検討を行った。
さらに,抗原調製と供給の方法,抗体価測定キット開発,測定データの集計と配信方法に関して,共同開発を行うことが可能な企業(4社)を検索して,コンソーシアム設立の準備を行った。
これらの調整と相互の成果の報告のため,岡山大学において本研究班とコンソーシアム参加企業が集合して,班会議を開催した。この結果をもとに,研究代表者は,コンソーシアム参加企業の各社と,検査の実施上の技術的問題,データ解析の方法,検査の普及のための方策等,種々の問題点を検討した。
さらに,臨床検査関連の各種学会において,歯周病細菌に対する血清IgG抗体価の測定とその応用に関する歯科領域でのこれまでの成果を公表して,本検査の実施に際しての臨床検査学分野での問題点を洗い出した。
日本歯周病学会の研究委員会が主催する学会のワークショップにおいて途中までの成果を公表し,歯周病細菌に対する血清抗体価測定検査ために,本研究班を中心として基盤研究(A)を申請した。 -
Integrate research of genomics and proteomics of novel molecular targets for development of periodontal diseases cure
Grant number：16209063 2004 - 2007
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
ABIKO Yoshimitsu, KURIHARA Hidemi, MURAKAMI Shinya, NAKAYAMA Koji, AMANO Atsuo, TAKASHIBA Shogo
Grant amount：\49530000 （ Direct expense: \38100000 、 Indirect expense：\11430000 ）
Experimental research utilizing functional genomics technologies with DNA/protein database will serve to identification of genes and their expressions on the transcriptional level exemplify their utility for the understanding the life science. Several bacterial genome projects associated with oral infectious diseases are in progress. Human genome has been sequenced and assembled, making the accessible for genetic studies in near future. Recently, technological advances, such as subtractive gene cloning and DNA microarray, have been applied to discover of novel genes involved in complex metabolism. For example, since little is known regarding the molecules expressed by gingival epithelial cells that are involved in inflammation following the interaction with periodontal pathogens, to find transcriptional profiling of genes in human gingival epitherial cells in response to LPS, we examined many altered gene expressions using over 8,000 cDNA microarray (Incyte, GEM). To find transcriptional profiles of genes in submandibular gland by ageing, we examined mRNA levels of over 6,500 genes by nucleotide micreoarray (Affimetrix, GeneChip). Our laboratory involved in dental science projects using these technologies and genome database. By this Grant support, we plan to make the new database for oral diseases, which will be used easily by many researchers. The genome project and molecular biological approaches using the database will open the gate develop the dental science.
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Preparation of self-organized nano-structured apatite and the protein adsorption property
Grant number：16360330 2004 - 2006
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
HAYAKAWA Satoshi, OSAKA Akiyoshi, TSURU Kanji, YOSHIDA Yasuhiro, SUZUKI Kazuomi, TAKASHIBA Shogo
Grant amount：\15000000 （ Direct expense: \15000000 ）
The selective protein adsorption property and the local structure around carbonate ions of nanocrystalline hydroxy-carbonate apatite were examined. Considerable changes in the selectivity in the adsorption of BSA and β_2-MG were observed due to the incorporation of the carbonate ions in hydroxyapatite lattice. The chemical states of the incorporated carbonate ions were examined by the 2D NMR spectroscopy. At least four or five peaks assignable to carbonate ions in A-site(OH) and B-site(PO_4^3) were observed in 2D ^<13>C{^1H} or ^<31>P{^1H} Het-Cor NMR spectroscopy. The surface charge distribution and the decrement of polar groups such as OH groups due to the distribution of carbonate ions in both A-and B-sites of the hydroxyapatite lattice are particularly favorable for β_2-MG adsorption rather than for BSA adsorption
We prepared nano-crystalline Zn-containing hydroxyapatite (ZnHAp) by the wet-chemical method and examined the selective adsorption of essential proteins, taking bovine serum albumin (BSA) and pathogenic protein such as β_2-microglobulin (β_2-MG) as model proteins. The increase of Zn content led to smaller crystallites and their specific surface area of ZnHAps increased with increasing the Zn content, accordingly. Furthermore, the amounts of BSA adsorption on ZnHAp particles decreased with increasing the Zn content in spite of the increase in the specific surface area. As the Zn^<2+> ion content in the apatites increased, the adsorbed amount of BSA was almost constant, whereas that of β_2-MG increased -
A study of the healing mechanisms of destructive periapical lesions and regenerative medicine for periapical bone defect
Grant number：16209056 2004 - 2006
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
ANAN Hisashi, MAEDA Katsumasa, MAEDE Hidefumi, SHIMAUCHI Hidetoshi, TAKASHIBA Shogo, KAWASHIMA Nobuyuki
Grant amount：\47060000 （ Direct expense: \36200000 、 Indirect expense：\10860000 ）
It has been reported that there are three key factors, such as cells, scaffolds and signaling molecules (growth factors), in the regenerative medicine. However, regenerative mechanisms of large-size defects in periapical lesions are not yet well understood. This study presented herein was therefore undertaken to define the progression and healing mechanisms in periapical lesions, and the effects of regenerative materials was investigated. It was showed that transplanted proliferating tissue produced by GTR membrane promoted the formation of new periodontal ligament(PDL) around the tooth. We have established three immortal human PDL fibroblast line by transfecting SV40T-Ag and hTERT into primary PDL fibroblasts.These immortalized cells was useful models for elucidating the biological features and regenerative mechanisms of human PDL. Mineral Trioxide Aggregate could up-regulated osteopontin and osteocalcin mRNA in human PDL to induce their differentiation. FGF-2 enhanced hyaluronan production by both human dental pulp cell and human PDL cell and hyaluronan synthase (HAS)1 and HAS2mRNA expression in both cells. Immune and nervous systems play key roles in periapical pathosis, and functional interactions between antigen-presenting cells and nerve fibers may play some roles in the development of self-defense reactions in periapical lesions. In addition, expression of RANKL is correlated with periapical lesion expansion, and followed by the expressions of RANK and OPG. Platelet-rich plasma (PRP) and washed platelets were potent inhibitors of RANKL-induced osteoclast differentiation in RAW264.7 cells. After application of Emdogain containing enamel matrix protein to the root surface, the formation of new cementum and more remarkable recovery of the bone tissue were observed. Although the expression of cytokines, such as IL-1β, RANKL, and RANK were hardly seen, BMP-2and BMP-4 expressing macrophages were increased. It was suggested that wound healing macrophages may express BMP and play an important role in the regeneration of periodontal tissue at the apex in EMD application.
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Diagnostic approaches by a susceptibility determination based on gene polymorphism in Japanese periodontitis patients.
Grant number：16209062 2004 - 2006
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
NAGATA Toshihiko, YOSHIE Hiromasa, MURAKAMI Shinya, TAKASHIBA Shogo, KURIHARA Hidemi, IZUMI Yuichi
Grant amount：\50050000 （ Direct expense: \38500000 、 Indirect expense：\11550000 ）
Polymorphism analyses using the invader method were performed (case control studies). From 12 hospitals, 622 blood samples were collected : 172 aggressive periodontitis (AgP) and the 178 controls, and 147 chronic periodontitis (CP) and the 125 controls. When AgP and the controls was compared, significant differences were detected in FcaR56T/C and MMP-3(-1171)5A/6A(-/T). When CP and the controls was compared, significant differences were detected in IL-1(+4845)G/T. In these SNPs, there were no SNPs showing both differences concerning gene distribution and allele frequencies. Further examinations are necessary to clarify these points. On the other hand, several interesting results concerning SNPs were obtained from the investigator's laboratories as follows. 1) In gingival overgrowth patients, a2 integrin +807 polymorphism was found, indicating the +807C allele is one of the genetic risk factors for drug-induced gingival overgrowth. 2) The combination of stimulatory FcyIIA and inhibitory FcyRIIB genotypes may increase susceptibility to SLE and periodontitis in the Japanese population. 3) Salivary AST, ALT and LDH levels reflect inflammation and destruction of periodontal tissue, suggesting the clinical useful markers following periodontal therapy but IL-1A+4845 alleles may not influence clinical parameters. 4) The mutant (A115V) TNSALP gene produced the defective alkaline phosphatase enzyme and it could be recessively transmitted and be a disease-causing mutation of the adult-type hypophosphatasia 5) Mannnose-binding lectin (MBL) gene mutation and smoking would be involved in the excerbation of aggressive periodontitis, via gene-environmental interaction. (229 words)
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バイオフィルムにおける歯周病細菌病原因子発現様態のゲノム-プロテオミクス解析
Grant number：16659499 2004 - 2005
日本学術振興会 科学研究費助成事業 萌芽研究
苔口 進, 福井 一博, 高柴 正悟, 西村 英紀, 前田 博史, 狩山 玲子, 井上 哲圭
Grant amount：\3300000 （ Direct expense: \3300000 ）
本研究は難治性の口腔バイオフィルム感染症である歯周病に関して、どのような機序でバイオフィルムを形成し、抵抗性を獲得し、またその中で歯周病細菌が病原性を発揮するのかについて分子生物学的手法を用いて解明することを目的とした。今年度は以下のような研究実績の概要である。
1)歯周病細菌Porphyromonas gingivalisの増殖や膿瘍形成に与る可能性のある遺伝子としてribonucleotide reductase D遺伝子(nrdD)を特定した。またP.gingivalisのバイオフィルム形成の際機能する二成分情報伝達系による発現調節網の解析を行ない、新規二成分系転写調節因子をコードする遺伝子を特定した。nrdDおよび二成分系転写調節遺伝子の欠損株を作成し、現在バイオフィルム形成、蛋白発現、さらには遺伝子発現パターンを親株と比較検討している。
2)現在、歯周病病巣バイオフィルムの生息し歯周病との関わりが注目されている未知難培養細菌の歯周病巣における分布様態を調べた。重度な歯周炎病巣ほどメタン産生古細菌であるMeth anobrevibacter種の検出割合が高く、患者血清の中にはM.oralisおよびM.smithiiの菌体蛋白と反応するものも認められた。さらに宿主細胞や免疫担当細胞との反応性を調べ、病原因子の特定を進めている。
3)バイオフィルム実験モデル系として、ガラスキャピラリー中で細菌バイオフィルムを形成させ、蛍光染色キットを用いて生菌と死菌を染め分け、共焦点レーザー走査型顕微鏡を用いて観察するキャピラリーフローセルシステムを確立した。抗バイオフィルム効果測定の新しい実験・評価や抗バイオフィルム剤の探索にペグ付き96穴マイクロプレートを用いる系の有用性を確認した。この系でクランベリーなど天然物から新規抗バイオフィルム効果のある物質を見出そうとしている。 -
歯周靭帯細胞における機械的ストレス応答性遺伝子のグルーピングと転写機構
Grant number：16659579 2004
日本学術振興会 科学研究費助成事業 萌芽研究
明貝 文夫, 高柴 正悟, 西村 英紀, 新井 英雄
Grant amount：\2800000 （ Direct expense: \2800000 ）
【目的】
機械的刺激が培養ヒト歯根膜線維芽細胞(HPLF)に及ぼす遺伝子発現変化を,マイクロアレイを用いて網羅的に解析する。
【材料と方法】
1.HPLFの刺激およびRNAの抽出
HPLFに,Flexercell Strain Unitを用いて機械的刺激を0.5,1,2,16時間与え,全RNAを回収した。刺激は1分間に6回の割合に5秒間ずつの緊張と弛緩を繰り返し行った。刺激を与えないものをコントロールとした。
2.マイクロアレイ解析
機械的刺激が細胞に及ぼす遺伝子発現変化を,Human Genome Focus Array(Affymetrix;約8,500遺伝子)を用いて,標的遺伝子のmRNA発現量を調べた。統計的手法を用いて解析した。
3.発現を変化する標的遺伝子の抽出とその機能
機械的刺激による発現量の変化が2倍以上を示した標的遺伝子として抽出した。それらの既知の機能をGeneSpring databases(Silicone Genetics)で調べ,系統別にカテゴリ分類した。
【結果】
1.標的遺伝子の抽出とその機能
機械的刺激によってその発現量が2倍以上変化するものは122であった。これらの標的遺伝子は,発現動態から8つのクラスターに分けられた。全てのクラスターはCell Growth and Maintenanceカテゴリ,あるいはIntracellular Signalingカテゴリに属する遺伝子を含んでいた。遺伝子の発現動態とcategoryに関係を見つけることができなかった。しかしながら,各々のクラスターは,Intracellular SignalingあるいはCell Surface Linked Signal Transductionカテゴリに属する遺伝子を含んでいた。
【考察と結論】
HPLFにおいて,機械的刺激は,外界からの刺激を感知しそして細胞内へシグナルとして伝える分子だけではなく,細胞増殖や代謝に関わる分子の発現に役割を果たすと考えられる。 -
X-ray Crystal Structural Analysis of Bacterial Iron Binding Protein and Development for New Antibiotics
Grant number：15390566 2003 - 2006
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
KOKEGUCHI Susumu, FUKUI Kazuhiro, TAKASHIBA Shogo, NISHIMURA Fusanori, MAEDA Hiroshi, ARAI Hideo
Grant amount：\12600000 （ Direct expense: \12600000 ）
In this study, we investigated the X-ray structural analyses on the iron-binding proteins(Dps (DNA protection during starvation) protein and ferritin protein) from peridontopathic bacteria, Actinobacillus actinomycetemcomitans, which play an important role in protecting cellular macromolecules from damage by reactive oxygen species.
A.actinomycetemcomitans expressed two ferritin protein(Ftn1 and Ftn2). The Ftn 1 could be successfully crystallized, but Ftn 2 became only amorphous form. Crystals of the Dps-like protein of A. actinomycetemcomitans were grown by the hanging drop method of vapour diffusion from a solution containing 28% polyethylene glycol 400 in 0.1 M HEPES-Na buffer pH 7.5 with 0.2 M calcium chloride dihydrate. Small but perfect hexagonal rods were grown with a protein concentration of~10 mg/ml. These rods diffract strongly to 1.9 A in-house and were found to be in the hexagonal space group P63 with cell dimensions a = b = 128.5 A, c = 91.lA and γ=120°. A 93.9% complete data set was collected to 1.9 A with a multiplicity of 2.9 and an R_<merge> of 0.071. The structure of the Dps-like protein of A.actinomycetemcomitans was solved by molecular replacement using the dodecameric ferritin like protein of Listeria innocua as the search model. The asymmetric unit contains four unique subunits arranged around the crystallographic 3-fold axis.
We also analyzed the antimicrobial activity against oral bacteria of human beta-defensin-2 (hBD-2) as a candidate of new antimicrobial substances. hBD-2 shouwed antimicrobial activity gainst Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, which was approximately equal to that of minocycline at equimolar concentrations. And we further examined the effect of ingredients and urinary metabolites of cranberry, which is rich in polyphenols, has been found to have various effects beneficial to human health, on biofilm formation by Escherichia coli. We found that ferulic acid, homovanillic acid, 4-coumaric acid, isoferulic acid and vanillic acid with inhibitory activity on Escherichia coli biofilm formation. -
口腔インプラントの骨結合獲得難易度を予測する生物学的診断法の開発
Grant number：15659463 2003 - 2004
日本学術振興会 科学研究費助成事業 萌芽研究
窪木 拓男, 高柴 正悟, 滝川 正春, 荒川 光, 藤沢 拓生
Grant amount：\3300000 （ Direct expense: \3300000 ）
1.チタンの細胞培養および遺伝子発現への影響
骨芽細胞様細胞株(MC3T3-E1細胞)の細胞培養培養および遺伝子発現に対するチタンの影響を検討した。
1)チタンプレート
ポリスチレン製の培養皿と表面粗さを同程度にするために,研磨ガラスにチタンを真空蒸着したものを使用した。
2)細胞接着への影響
通常の培養皿と比較してチタンは細胞接着を抑制する傾向にあった。
3)細胞増殖への影響
通常の培養皿と比較して,細胞播種後1,2日ではチタンでは増殖が抑制されるものの3日では両材料ともコンフルエントに達した。
4)細胞分化への影響
骨芽細胞の分化の指標のひとつであるアルカリホスファターゼ活性は,両材料ともに細胞がコンフルエントになった後5日目ごろより上昇し,14日目でピークを向え,21日目では低下した。チタンでは通常の培養皿と比べてアルカリホスファターゼ活性は抑制された。
5)遺伝子発現への影響
通常の培養皿と比較し,チタンの遺伝子発現への影響をサブトラクティブハイブリダイゼーション法にて検討したところ,両材料間で発現に差のあるsod-1,xab-2の遺伝子を検出した。
6)リアルタイムPCR法による遺伝子発現の変動
サブトラクティブハイブリダイゼーション法にて検出した発現に差のあるsod-1,xab-2の経時的な発現の変動を検討したところ,培養皿では細胞播種後5日目で発現のピークを向え,その後低下した。チタンでは発現のピークが10日目前後と培養皿より遅延し,発現も抑制されていた。 -
日本人の腎結石から分離した新種ナノバクテリアに関する多面的解析
Grant number：15659381 2003 - 2004
日本学術振興会 科学研究費助成事業 萌芽研究
公文 裕巳, 門田 晃一, 筒井 研, 八木 直人, 高柴 正悟
Grant amount：\3300000 （ Direct expense: \3300000 ）
岡山大学泌尿器科で採取された日本人の尿路結石47検体、パラグアイで採取された尿路結石18検体、岡山大学一般歯科診療室で採取された歯石14検体を対象としてNanobacteria-like organism(NLO)の電子顕微鏡による観察、ならびに、分離培養を試みた。日本人の尿路結石とパラグアイ人の尿路結石でのNLOの検出率は、それぞれ61.7%(29/47)、66.7%(12/18)とほぼ同率であった。分離培養はそれぞれ7例と3例に可能であった。結石成分分析において、リン酸カルシウム含有率はNLO検出例約70%、分離培養例約78%と高率であったが、分析上でリン酸カルシウムを含有しないものからも検出・分離された。なお、歯石からは検出されなかった。
SPring-8での解析では、NLOの大きさが分解能以下のサイズであったにもかかわらず、アパタイト層の構築様式の三次元的解析が可能であり、個々のアパタイの外皮で被われたNLOが集簇的に融合、その集合体全体を包み込むように最外層にアパタイト層が構築されて成長することが明らかとなった。増殖培地の工夫により増殖様式はアパタイト型のほかに浮遊型が存在すること、その増殖速度も培養条件に左右されること、ならびにOD650でモニタリング可能であることが判明した。モノクローナル抗体で特異的に染色可能であることも明らかとなったが、Nanobacteriaに特異的であるとされたプライマー(フィンランドのグループのオリジナル文献:(Proc.Natl.Acad.Sci.USA,95:8274,1998)ならびに細菌属に共通のユニバーサルプライマーを用いるPCRでは特異的な反応は得られなかった。NLOの増殖のメカニズムに未だ不明な点が少なくないが、尿路結石等の異所性石灰化にNLOが関与することが強く示唆された。 -
Gene profiling of periodontal pathogens in periodontal lesion
Grant number：15592187 2003 - 2004
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
ARAI Hideo, MAEDA Hiroshi, KOKEGUCHI Susumu, TAKASHIBA Shogo
Grant amount：\3300000 （ Direct expense: \3300000 ）
1.Host-induced genes of A.actinomycetemcomitans
Actinobacillus actinomycetemcomitans possess many virulence factors. However, the potential roles of the virulence factors are not well characterized. In addition, many unknown virulence factors are considered to be involved in the pathogenesis. A.actinomycetemcomitans grows as a rough colony on primary isolates (R-type). The colony converts to a smooth phenotype (S-type) through repeated subculture. Since the phenotypic alteration reflects different gene expressions in response to environmental changes from the in vivo to the in vitro, isolation of genes induced in the R-type strain turns out to be an isolation of host-induced genes, including the virulence factors. In the current study, we identified genes induced in the R-type of A.actinomycetemcomitans using the cDNA subtractive hybridization technique.
Three genes, mip,prx and ompA were identified as R-type specific genes. Attention was focused on the mip, and a recombinant Mip-like protein and the deficient mutant were created. The recombinant protein reacted with the patients sera, suggesting the production of the Mip-like protein in periodontal lesions. The mutant was used for an invasion assay and demonstrated impaired ability for the invasion of the host cells. The expression of the mip, prx and ompA may be enhanced in the host, and the Mip may play an important role for invasion of A.actinomycetemcomitans.
2.New system for microbiological examination.
Microflora of subgingival plaque is a very complicated community. Therefore, microbiological examination is required for the analysis of periodontal pathogens and virulence factors in vivo. In the current study, we established a new system for the examination. Loop-mediated isothermal amplification (LAMP) method was employed for the system. Primers were designed from the nucleotide sequence of 16S ribosomal RNA gene, and the LAMP method detected periodontal pathogens with high sensitivity, specificity and rapidity. -
Regulation of gene expression of periodontal virulence factors during biofilm formation
Grant number：15591932 2003 - 2004
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
INOUE Tetsuyoshi, SHINGAKI Ryuji, KOKEGUCHI Susumu, TAKASHIBA Shogo, FUKUI Kazuhiro, OHTA Hiroyuki
Grant amount：\2700000 （ Direct expense: \2700000 ）
<Observation of biofilm (BF) formation> In a wild-type strain of Actinobacillus actinomycetemcomitans (Aa), many fimbriae were produced in an early stage of BF formation, whereas, in mature stage, fimbriae production was repressed, and cell-cell aggregation was observed. Exopolysaccharide (EPS)-like structure was also observed on the cell surface. <BF formation and dye-binding ability> BF-positive strains had Congo red (CR)-binding ability, while BF-negative strains did not. A fimbriae-deficient strain showed CR-binding ability, suggesting that it indicates the presence of BF formation factors other than fimbriae. The dye-binding capacity disappeared by treatment with periodate, suggesting it reflects EPS biosynthesis. From genome sequence analysis, a gene cluster homolog involved in biosynthesis of Congo red-binding EPS was found in Aa genome. One of the genes was disrupted. However, BF formation was not affected. More detailed studies are needed to clarify the role of this gene cluster in BF formation. <Assay for cell-cell aggregation> Treatment with periodate or DNase completely inhibited cell-cell aggregation. From this result, it was assumed that in addtion of EPS, cell surface DNA was also involved in aggregation during BF formation. <Regulation of leukotoxin production> To examine the effects of catabolite repression-like mechanism on BF formation and leukotoxin production, attempts to isolate crp gene mutant were made. However, it colud not be obtained. The crp gene might be an essential gene in Aa. Toxin production was increased in an acidic condition, suggesting the reduction of microenvironmental pH in BF causes induction of toxin production.
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Gene Therapy for Periodontal Diseases -Regulation of host response by local gene delivery-
Grant number：14370710 2002 - 2005
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
TAKASHIBA Shogo, KUBOKI Takuo, NISHIMURA Fusanori, KUBOTA Satoshi, MYOKAI Fumio
Grant amount：\13500000 （ Direct expense: \13500000 ）
Because understanding the target factor was important, we decided to specify the target factor from both sides of non-specific host defense and tissue regeneration. Moreover, because it was also anxiety against clinical application of gene therapy because of its toxicity, the experiments are performed for testing it.
1.Target gene
In the rat, cytochrome c oxidase gene expressed strongly at 1 week and pro-α-2 type I collagen gene expressed strongly at 2.5 weeks after alveolar bone begun regeneration. Activation of these genes seem to be needed for alveolar bone regeneration. In dental pulp would, the homolong of human 12.7K-interacting protein 2 expressed strongly. We named it rat FIP-2 gene. It is under analyzing now for physiological meaning. In addition, inflammation, promoter region required for LPS-induced transcription of LITAF was revealed, which is new transcription factor for human tumor necrosis factor(TNF)-α.
2.Introduction of β-defensin by non-viral vector
Anti-bacterium peptide β-defensin gene was transferred to human epithelial cells and rat salivary glands to test its effect for reduce bacteria around cultured cells or rat oral cavity. Furthermore, its effect for local tissue inflammation was also examined. We found that bacterial numbers are reduced and that no obvious inflammation in rat salivary gland tissue. However, when electroporation was used for gene delivery, obvious inflammation was detected. Furthermore, It was revealed that β-defensin gene transcription requires 2 regions of NF-kB binding domain on its promoter, but that NF-IL6 biding domain on it acts to reduce its transcription. -
Establishment of a autologous cell transplantion method using mesenchymal stem cells for perio-dontal tissue and alveolus bone regeneration.
Grant number：14370632 2002 - 2004
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
KUBOKI Takuo, UEDA Minoru, TAKIGAWA Masaharu, TAKASHIBA Shogo, MAEKAWA Kenji, YOSHIDA Yasuhiro
Grant amount：\14800000 （ Direct expense: \14800000 ）
1.Isolation human bone marrow cells and cell culture.
Bone marrow cells were isolated from human iliac bone marrow of volunteer. Human bone marrow cells were plated and cultured in DMEM containing 10% FBS. Culture medium was changed every 3 days, and their multipotent (osteoblastic and adipogenic) differentation abilities were confirmed
2.Connective tissue growth factor (CTGF/CCN2) enhanced hMSC attachment, migration and survival in a hydroxyapatite scaffold
Human bone marrow cells were incubated to attach onto porous HA blocks for a week. The porous HA/cells hybrids were implanted subcutaneously in nude mice (4 week-old) with CTGF (1ug) or distilled water (control).
The implants were harvested after 4 weeks for SEM observation. SEM observation supported that hBMSC-like cells migrated and survived inside of the porous HA scaffold with CTGF application, while without CTGF, no viable cells were observed inside of the scaffold.
3.hMSC initial attachment and proliferation enhanced on titanium
Adsorption of poly phosphoric acid to Ti disk surface was achieved by immersing Ti disk poly phosphoric acid solution (1 wt%) for 24 hours. Adsorption of polyphosphoric acid onto Ti disks enhanced attachment and proliferation of hMSC.
4.Effect of gene expression of osteoblast on titanium
Osteoblast was cultured on titanium dish and searched a titanium specific gene by cDNA subtractive hybridization. It became clear on titanium that sod-1, gene expression of ribosomal protein L19 were restrained significantly. -
歯周病原性細菌によって起こる誤嚥性肺炎の分子免疫学的病態の研究
Grant number：14657554 2002 - 2003
日本学術振興会 科学研究費助成事業 萌芽研究
高柴 正悟, 前田 博史, 明貝 文夫, 苔口 進
Grant amount：\2600000 （ Direct expense: \2600000 ）
誤嚥性肺炎は,食物や口腔内細菌などの誤嚥に起因する肺炎であり,主に高齢者に発症する。とりわけ歯周病に罹患している高齢者の口腔内には大量の歯周病細菌が存在するので,誤嚥性肺炎発症のリスクは高いと考えられる。また,肺炎は重篤になると肺局所の炎症にとどまらず,全身症状の悪化をきたし時に死を招くこともある。したがって誤嚥性肺炎を発症した肺局所の炎症巣の全身に対する影響を知ることは重要である。我々は,本研究援助の下,(1)誤嚥性肺炎のマウスモデルを構築し,(2)肺局所と血清中のIL-1β,IL-6,TNF-α,そしてTNF-αのアンタゴニストである可溶性TNF受容体(sTNFR1およびsTNFR2)の産生動態を比較検討した。誤嚥性肺炎のマウスモデルは,代表的な歯周病細菌であるPorphyromonas gingivalis(P.g)の死菌体をマウスの肺に直接,感染させて構築した。このモデルは,組織学的に,P.g感染後,1-3日後の肺に著明な炎症性細胞浸潤を認め,7日後の肺では健常レベル回復するという比較的弱い炎症症状をきたすものである。我々は,この肺炎マウスにおける肺と血清中において,各種サイトカイン産生量を経時的(感染後2時間,1,3,7日)に測定した。IL-1βは,肺局所において感染後2時間-3日後に有意に増加したが血清中では検出できなかった。IL-6は,肺局所において感染後2時間-3日後に有意に増加し,血清中においても感染後2時間で有意に増加した。TNF-αは,肺局所において感染後2時間のみで有意に増加したが,血清中では検出できなかった。またsTNFR1の産生量は感染の有無によって,肺局所,血清中ともに変化しなかった。一方,sTNFR2は肺局所において感染後2時間-3日後に有意に増加したが,血清中では変化しなかった。また,感染後2時間で血清中のsTNFR2/sTNFR1比が有意に増加した。以上の結果から,この血清中におけるIL-6とsTNFR2の産生量の増加が肺の局所炎症に対する全身反応であることが示唆される。この研究結果により,将来の局所炎症に対するサイトカインを用いた炎症制御療法の発展が期待される。
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Role of a novel transcription factor for TNF-α, LITAF, on the pathogenesis of periodontitis
Grant number：14571982 2002 - 2003
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
MYOKAI Fumio, ARAI Hideo, NISHIMURA Fusanori, TAKASHIBA Shogo, KOHNO Takayuki, MAEDA Hiroshi
Grant amount：\4000000 （ Direct expense: \4000000 ）
LPS-induced TNF-α factor (LITAF) is a recently identified novel transcription factor controlling TNF-α gene expression in human monocytic cells (Myokai et al., Proc Natl Acad Sci USA, 96: 4518-4523, 1999). To characterize LITAF promoter, we isolated a 1.2 kb fragment of human genomic DNA containing 5'-flanking sequence of the LITAF gene. Primer extension analysis revealed that a transcription start site situated 234 bases upstream from the translation start site in the LITAF gene. The promoter sequence contained the similar consensus sequences for AP-1 and NF-IL6 which were known to be responsible for signaling by LPS. For reporter gene assays in human T-cell leukemia cell line (Jurkat), a series of constructs with fragments of increasing length of the LITAF promoter were coupled. to the firefly luciferase gene. A 34-bp sequence domain located from nucleotides -76 to -43 in the promoter, in which the consensus binding site for AP-1 and NF-IL6 was missing, exhibited the highest reporter gene activity. Moreover, the domain did not include any known consensus sequences. These results suggest that the new sequence domain contributes the up-regulation of LITAF gene transcription.
In the following study, we aimed to examine the localization and kinetics of LITAF protein in THP-1 cells stimulated with LPS. By immunological staining using anti-LITAF monoclonal antibody, an accumulation of LITAF protein was detected in the nuclei of the LPS-stimulated cells whereas it was detected in the cytoplasm of static control cells. Western blot analysis revealed the kinetics of protein in both nuclei and cytoplasm of THP-1 cells stimulated with or without LPS. The nuclear LITAF protein was increased followed by 2 h stimulation, whereas it was recovered its basal level even by the stimulation between 2 and 24 h (student t test; p<0.05). No significant change in the cytoplasmic LITAF protein level was detected followed by the stimulation between 0.5 and 24 h. These results suggested that LITAF protein was transported form the cytoplasm to the nuclei by in the LPS stimulation. Some DNA binding proteins may serve the transportation of the LITAF protein, because the LITAF protein is lack of nuclear localization signal. -
Development of the methods for application of the factors that biologically accelerate reparative dentin formation
Grant number：14571844 2002 - 2003
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
SONOYAMA Wataru, TAKIGAWA Masaharu, TAKASHIBA Shougo, KUBOKI Takuo
Grant amount：\3900000 （ Direct expense: \3900000 ）
1. Pulp cell isolation from human extracted tooth and confirmation of their phenotype
Under permission of ethical committee, pulp cells were isolated from human extracted tooth. RT-PCR was carried our to confirm their gene expression profile and phenotype. As a result, they expressed odont oblast-specific gene, dentin sialophosphoprotein (DSPP), and were suspected to be odont oblast lineage.
2. Effects of Growth factors on their attachment, proliferation, and differentiation
Effects of growth factors, e.g., transforming growth factor-beta1 (TGF-beta1), basic fibroblast growth factor (bFGF), and connective tissue growth factor (CTGF), on their attachment to plastic dish were investigated. As a result, adsorption of these growth factors enhanced their attachment to plastic dish. Effects on their proliferation and alkaline phosphatase (ALPase) activity were also investigated. Concerning about proliferation, only TGF-beta1 enhanced their proliferation. While ALPase activity was downregulated by TGF-beta1 and bFGF.
3. Effects of hydroxyapatite (HAP) on their attachment
To estimate compatibility of HAP with pulp-derived cells, their attachment onto HAP was investigated. As a result, attachment onto HAP was significantly higher compared to plastic dish made of polystyrene. Adsorption of growth factors onto HAP tended to enhance attachment, but the difference was not significant.
4. Effect of HAP on their gene expression
To estimate that HAP have an effects on gene expression of pulp-derived cells or not, they were seeded onto HAP and their gene expression were investigated by RT-PCR. As a result, DSPP and type 1 collagen gene expression were significantly enhanced. -
歯周組織の治癒に関わる遺伝子の研究
Grant number：01F00761 2001 - 2002
日本学術振興会 科学研究費助成事業 特別研究員奨励費
高柴 正悟, 村山 洋二, PETELIN Milan, PETELIN M.
Grant amount：\1000000 （ Direct expense: \1000000 ）
研究1 歯周病細菌Porphyromonas gingivalis (Pg)感染した肺炎マウスにおける局所・全身のTNF-αおよび可溶性TNFレセプターの産生動態
近年,歯周病細菌が何らかの経路で遠隔臓器に感染し,様々な為善作用を及ぼすことが知られるようになってきた。しかし,その詳細な生体反応のメカニズムについては不明である。本研究は,代表的な炎症性サイトカインであるTNF-αおよび可溶性TNFレセプター(sTNFR)の産生動態を指標に,Pg感染性肺炎が全身にどのような影響を及ぼすのかを調べた。
【方法】Pg感染を肺炎マウスにおいて,経時的にその肺抽出液および血清中のTNF-αおよび可溶性TNFレセプターの産生量を市販のELISAキットを用いて調べた。
【結果】肺抽出液中:TNF-α量は,Pg感染後2時間で有意に高い値を示した。また1型sTNFRの産生量は,感染の有無に関わらず変化しなかったが,2型sTNFRの産生量は,感染後1-3日まで有意に高い値を示した。血清中:TNF-α量は,感染の有無に関わらず変化しなかった。また,2型sTNFR/1型sTNFR比は,Pg感染後2時間で有意に高い値を示した。
【考察および結論】Pg感染後,肺局所において産生されたTNF-αの為害作用は,局所・全身ともに2型sTNFRの産生量が増すことによって抑制制御されている可能性がある。したがって,TNF-αを中心とした局所炎症の拡がりを制御するには,2型sTNFRが有効なのかもしれない。
研究2 ラット唾液腺に発現させた抗菌ペプチドを用いた歯周病抗菌療法における基礎研究
近年,医科額域における遺伝子治療の発展は目覚ましい。この流れから,我々は歯周病における遺伝子治療の応用を検討している。本研究では,ラット唾液腺に遺伝子を強制発現するための有効な手段を探るため,効率のよい遺伝子導入法を検討した。
【方法】ラット唾液腺に電気的手法,化学的手法および直接法の3種遺伝子導入方法でβ-gal遺伝子を発現させ,その発現強度を比較検討した。
【結果】化学的手法による遺伝子導入方法が,他の手法に比して有意に高いレベルでβ-galを発現した。
【考察および結論】歯周病抗菌療法には,化学的手法を用いた遺伝子導入法が有効な手段であることを示唆する。今後,βデフェンシンなどの抗菌物質を唾液腺に強制発現させ,歯周病抗菌療法の応用に向けた有効性を検討する予定である。 -
Project study of tissue regeneration with tissue engineering
Grant number：12307051 2000 - 2003
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
YAMADA Satoru, MAEDA Katumasa, TAKASHIBA Shogo, KURIHARA Eiji, ODA Shigeru, HASEGAWA Kouji
Grant amount：\39730000 （ Direct expense: \33700000 、 Indirect expense：\6030000 ）
1. Mesenchymal stem cells : Proliferating tissue in periodontal osseous defects promotes the formation of new periodontal tissue around the root(Yamada.Ota). We find that the extracellular matrix is a critical regulator of the induction of alkaline phosphatase and the formastion of multiple layered in human gingival fibroblast(Maeda). One clone, PDL-29, identified as a COX assembly factor, showed much stronger m RNA expression in HPEs than in HGFs in culture. (Takashiba)
2. Signaling molecules :
Placement of PGS between the root surface and rhBMO-2 3 PGS complex had the effect to decrease ankylosis(Kawanami). SPARC plays arole in repair of periodontal tissues by promotingproliferation and osteoclastogenesis inhibitory factor production(Kurihara). EMDOGAIN has a potential to promote the cementum regeneration and to create a favorable environment that will promote the periodontal regeneration. CPC seemed to act as a scaffold for bone formation and histocompatible healing of periodontal tissues(Oda). PGE2, cAMP-elevating agent, EP2/EP4 agonist stimulated cAMP accumulation in HPDL cells. However, BMP-2 hand no effect on it, and per-treatment with BMP-2 also did not cause significant changes in the cAMP accumulation stimulated with PGE2 or EP2 / EP4 agonist(Hasegawa). LJE becomes shorter, whereas the proliferative activity of regenerative connective tissue maintains the same level of proliferation, and ultimately LJE is replaced by regenerative connective tissue(Hashimoto9.Topical application of 0.1-0.4% bFGF induced significant periodontal tissue regeneration in animal experiments. In vitro studies demonstrated that bFGF stimulation in the presence of fetal calf serum inhibited proliferation of gingival epithelial cells and induced the release of hyaluronan and heparan sulfate from periodontal ligament cells (Murakami)
3. Scaffolds : The PLLA membrane showed an excellent results in comparison with the PLGA membrane in vitro experiment. Moreover, the histological observation showed no different bone regeneration in dogs in between PLLA and PLGA membrane(Izumi). The effect of matrix geometry upon the periodontal reconstruction induced by BMP was studied. This study using geometrically different cell substrata demonstrated that a matrix with a certain geometrical size is most favorable for cell differentiation((Takida) -
Study on the influence of periapical lesions on systemic health
Grant number：12307044 2000 - 2002
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
ISHIHARA Sachiyo, KAWASHIMA Nobuyuki, NOIRI Yuichiro, TAKAHASHI Keiso, HASEGAWA Masako, HAYASHI Yoshihiko
Grant amount：\34980000 （ Direct expense: \30600000 、 Indirect expense：\4380000 ）
Anaero bic bacteria in infected root canals and the biofilm produced by the bacteria have been analyzed biochemically and micro morphologically. The activity forming the biofilm differed among bacteria examined and glycocalyx-like concentration has been found outside the biofilm. We have established the methodology of isolation and identification for E. faecalis by chair-side anaerobic culture system. In addition, a novel identification method for bacteria in infected root canals by polymerase chain reaction has been developed. Relationship between systemic diseases and periapical lesions has been investigated on various types of compromised hosts, such as non-Hodgkin's, hepatitis C, refractory skin diseases and the aged patients over 70 years old. No significant relationship of the changes of CRP values after infected root canal treatment was found. We have investigated the influence of oral infection in the pathogenesis of Pustulosis Palmartis et Plantaris (PPP), by measuring the serum titer of Interleukin-8 (IL-8). Although the titer of IL-8 in PPP patients accompanied with periodontal and/or periapical lesions was not significantly higher than that in healthy subjects, the adult atopic dermatitis patients accompanied with the legions, up-regulation of serum IL-8 was induced by endodontic or periodontal treatments which would cause bacteriemia in PPP patients. Improvement of clinical symptoms of PPP was obvious after the treatments. These results indicated that the serum IL-8 was susceptible to bacterial infection in the PPP patients, and overproduction of IL-8 might modify the initiation and progress of PPP. Most of aged person tended to suffer from periodontal disease in addition to periapical lesions and then we need to collect the subjects whom we can evaluate the influence of periapical lesions alone. We have performed root canal therapy to the patient who has 20 infected root canals and shows compromised according to using adrenocortical hormones. The degree of CRP in the serum of this patient decreased to normal levels, although tic therapy did not.
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Molecular cloning of the factors that biologically accelerate reparative dentin formation
Grant number：12470418 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
KUBOKI Takuo, TAKIGAWA Masaharu, TAKASHIBA Shougo, SONOYAMA Wataru, KANYAMA Manabu, NAKANISHI Tohru
Grant amount：\13800000 （ Direct expense: \13800000 ）
1. Gene delivery to cultured cells
We prepared recombinant adenovirus vector carrying beta-galactosidase gene. Mouse osteoblast-like cells, MC-3T3 -E1, were cultured with this vector. As a result, almost all cells were transfected with beta-galactosidase gene.
2. Localization of known factors (TGF-beta 1 and CTGF) in vivo animal model
Localization of TGF-beta 1, that is supposed to be involved in reparative dentinogenesis, and CTGF were confirmed with immunohistochemical staining in animal (wister rat) experimental model. As a result, in 2 weeks from tooth reduction reparative dentin-like tissues were observed, and strong staining of TGF-beta 1 and CTGF were observed around these tissues.
3. Gene expression of TGF-beta 1 and CTGF in cultured cells stimulated with proinflammatory factors
MDPC-23, mouse-derived odontoblast-like cells were used in this study. The cells were stimulated with IL-1 beta and bacterial LPS. Changes in CTGF and TGF-b1 genes expression were examined by RT-PCR. As a result, MDPC-23 cells were expressing the CTGF and TGF-beta 1 genes coastitutively, and both factors increased CTGF gene expression and decreased TGF-b1 gene expression in the odontoblast-like cells (MDPC-23) within one-day period after stimulation.
4. Effect of TGF-beta 1 and CTGF to cultured cells
The effects of rCTGF and rTGF-beta 1 on cell proliferation were determined by the MTT assay. rTGF-beta 1 tended to decrease the proliferation dose-dependently, whereas effect of rCTGF was not evident. Next, to investigate the effects of rCTGF on calcification of MDPC-23 cells, cells were cultured with medium containing rCTGF. Sequential addition of AA and b-GP up-regulated the ALPase activity, while addition of rCTGF had no obvious effects. -
Microbiological examination for periodontal therapy
Grant number：12557192 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
MURAYAMA Yoji, NISHIMURA Fusanori, ARAI Hideo, TAKASHIBA Shogo, KOKEGUCHI Susumu
Grant amount：\12300000 （ Direct expense: \12300000 ）
Real time PCR using GeneAmp^R sequence detection system; was applied to the microbiological examination in periodontal disease. Both TaqMan probe with reporter and quencher dye and SYBR Green dye were used for the source of fluorescence. The primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gigngivalis, Prevotella intermedia and total bacteria based on the nucleotide sequence of 16S ribosomal RNA gene. Since spread of antibiotic-resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confer resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans were linear over a range of 10 to 10^7 cells (10 to 10^7 copies for tetQ gene), while the quantitative range for total bacteria was from 10^2 to 10^7. There was no significant difference between the TaqMan and SYBR-Green chemistry systems in their specificity, quantitativity and sensitivity. The SYBR Green chemistry was then used for the clinical plaque samples. Subgingival plaque samples were obtained before and one week after the local drug delivery of minocycline. Although the number of P. gngivalis, P. intermedia and A. actinomycetemcomitans decreased after the antibiotic therapy in most cases, the plaque samples contained higher level of tetQ gene. The quantitative real time PCR will be a powerful tool for microbiological examination of periodontal disease and the quantitative monitoring of antibiotic resistance gene is thought to be necessary for periodontal therapy.
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歯周病の発症と進行に関わる交叉免疫応答を誘導する抗原蛋白
Grant number：12877343 2000 - 2001
日本学術振興会 科学研究費助成事業 萌芽的研究
村山 洋二, 大山 秀樹, 西村 英紀, 高柴 正悟, 河野 隆幸
Grant amount：\2000000 （ Direct expense: \2000000 ）
Prophyromonas gingivalis由来の分子量53kDaの外膜蛋白(Ag53)は,多くの歯周炎患者由来のIgG抗体およびヘルパーT細胞株によって共通して認識される領域(Ag53p141-161)を有する。本研究において我々は、歯周病細菌抗原由来蛋白を認識するT細胞が他の抗原蛋白と交叉応答し得ることを明らかにすることを目的とした。昨年度我々は、早期発症型歯周炎患者の末梢血単核球からAg53p141-161をHLA-DRB1^*1501拘束的に認識するTh細胞クローン(HT8.3)を樹立した。さらに,Ag53p141-161のアミノ酸配列と相同性を示す領域を有する蛋白5種類をデータベースから探り当てた。しかし,相同性を示す領域の合成ペプチドを作成したが,これらペプチドに対してHT8.3は応答性を示さなかった。このことは,Ag53p141-161の領域において,どの部位がT細胞の抗原認識に関わるかについての詳細を明らかにすることが必須であることを示唆するものである。
以上のことから本年度は,1)HT8.3の抗原認識に関わる詳細な部分を知ること,さらには2)Ag53p141-161において抗原認識に関わる詳細な部分のアミノ酸置換ペプチドに対するTh細胞の応答性を調べることを行なった。その結果,1)Ag53p141-161において,T細胞の抗原認識に関わる領域はAg53p144-155であること,2)Ag53p144-155においてp147(V)を1leにp151(A)をGlyにそれぞれ1残基置換したペプチドは,野性型ペプチドよりも低濃度でHT8-3の増殖応答を誘導することを突き止めた。
これらのペプチドの発見によって,歯周病細菌抗原由来蛋白を認識するT細胞が他の抗原蛋白と交叉応答し得ることの可能性,さらにはアナログペプチドを用いた免疫療法の進展への可能性が示唆された。 -
Periodontal Treatment by Regulation of Novel TNF-α Transcription Factor in Monocytes
Grant number：12470471 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
TAKASHIBA Shogo, MAEDA Hiroshi, MYOKAI Fumio, NISHIMURA Fusanori
Grant amount：\13100000 （ Direct expense: \13100000 ）
Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor alpha (TNF-α). It gives the deleterious effects to the host of TNF-α, especially advance of inflammation, the destruction of the tissue. It also gives same effects in the periodontal tissue.
We tried to control TNF-α gene transcription not using NF-κB strongly concerned with other activities of the cells but using LITAF (LPS-induced TNF-α factor) which we identified as a new transcription factor of its transcription. We found that inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-α transcription.
We could find strong LITAF promoter region by LPS stimulation and same consensus sequences as NF-IL6 and AP-1 in this region. On the other hand, we found LITAF localization because staining for LITAF was present in the nuclei of THP-1 after LPS stimulation. We also established gene transfection to find the best method for transfection in vivo.
In addition, we transfected mutants of 1κB-α which is one of the inhibitor of NF-κB into the THP-1 cells. We established only the sysyem of THP-1 cells without NF-κB influence, however we will discussed the influence of TNF-α expression by LITAF and the effect of LITAF without NF-κB even though this research period was over. -
Factor for induction of root resorption
Grant number：12671851 2000 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
MYOKAI Fumio, NISHIMURA Fusanori, TAKASHIBA Shogo, MURAYAMA Yoji, KOHNO Takayuki
Grant amount：\3500000 （ Direct expense: \3500000 ）
Using subtractive hybridization, we isolated genes from human periodontal ligament (PDL) cells that were differentially expressed in response to mechanical stress. MRG X gene which related to morality factor 4 gene was cloned from a human cDNA library by subsequent screening. It belongs to a novel family of transcription factors regulating cellular proliferation and senescence. Cellular proliferation is initiated by growth factor binding to specific receptors to initiate signal transduction. Transcripts for the MRG X gene were increased following mechanical stress in cultured PDL cells. The target genes for the MRG X are unknown. Transforming growth factor (TGF)-β1 is mitogenic to PDL cells and regulates the osteoblast-like phenotype of PDL cells. TGF-β1 and its type I receptor (TβR-I) genes were co-expressed following mechanical stress in cultured PDL cells. To examine the effects of the MRG X on the expression of TGF-β1 gene, we performed functional studies aimed at interfering with MRG X mRNA production in cultured PDL cells. Inhibition of MRG X gene mRNA expression in PDL cells resulted in transcription of both the TGF-β1 and TβR-I genes. An application of mechanical stress resulted in a marked reduction of the transcription of TGF-β1 gene in MRG X antisense-expressing cells. The expression level of endogenous MRGX mRNA was elevated by the mechanical stress in the cells. These findings suggest that MRG X acts as a negative regulator of transcription for both TGF-β1 gene in PDL cells.
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早期発症型歯周炎の病態解析と診断基準確立に向けた共同研究の企画調査
Grant number：12897021 2000
日本学術振興会 科学研究費助成事業 基盤研究(C)
山崎 和久, 高柴 正悟, 栗原 英見, 吉江 弘正, 相田 宜利, 村上 伸也
Grant amount：\3100000 （ Direct expense: \3100000 ）
早期発症型歯周炎(Early-Onset Periodontitis;EOP)は乳歯あるいは永久歯列を有する者において、いわゆる成人性歯周炎の発症時期よりも明らかに早期に発症し、その後、急速な進行経過をたどって歯の脱落にいたる疾患である。本邦における発症頻度は欧米におけるそれと比較してかなり低いといわれているが(岡本ら0.18%)、全国規模での疫学調査報告はなく、詳細については明らかになっていない。その発症には細菌学的、免疫学的要因の関与が示唆されているが、成人性歯周炎におけるそれらと同様、単一病原細菌による特異的な疾患ではなく、複数の細菌種が関与しているという報告がほとんどであるが、欧米においては主要な原因菌としてActinobacillus actinomycetemcomitans(Aa)がもっとも注目されており、その病原因子や、病態との関連について多くの報告がある。また、宿主防御機構の異常が示唆されることから免疫機能に関係している因子についても多くの研究があり、免疫担当細胞の機能異常との関連が示唆されている。しかし、日本人早期発症型歯周炎とAa菌の関連は低いとする報告や、宿主防御機能の低下についても統一された見解は得られていない。この理由は診断に統一された基準が無く、いずれもAAPの基準を参考に独自の基準を加えて行っていることによると思われる。そこで、本企画調査では日本人における早期発症型歯周炎の病態を明らかにし、診断の一助とするために患者集団の選択、細菌検査・免疫学的検査の項目・方法を統一するための基準作りをすることが決められた。
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Study for preservation of dentin using local gene deliveryof dentin-specific genes
Grant number：11557143 1999 - 2001
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
TAKASHIBA Shogo, MYOKAI Fumio, ARAI Hideo, MURAYAMA Yoji
Grant amount：\8700000 （ Direct expense: \8700000 ）
Genes related to dentin regeneration were isolated by susbtractive hybridization from rat molar dental pulp after dentin cavity preparation. There were 16 induced and 7 reduced genes found after size selection (more than 250 bp). Induced genes included cytochrome c, cathepsin B, 3 EST genes (unknown function), and 1 novel gene. Reduced genes included ribosomal protein, laminin-γ2, type 1 collagen-α2, and 2 novels genes. Although in situ hybridization analysis of these genes was performed in order to elucidate their expression sites, no clear results have not been obtained. However, Northem blot analysis revealed that 1 EST gene showed most different expression signals compared before and after cavity preparation. Full length cDNA of this gene was cloned and nucleotide sequence analysis and homology search were performed. It is 3.8-kb length and has 83% homology to human FIP-2 (Adinovirus 14..7 KDa - interacting protein) in some part. Furthermore, this gene has longer putative coding region than that of FIP-2, and posess coild-coild domain including zinc finger domain and leuicin zipper domain. On the other hand, local gene delivery to dental pulp was performed using plasmid vector and andenovirus-associate vector using reporter gene. No significant difference of transfection efficiency was found between these vectors because cells on only the surface of dental pulp were transfected. Primitive problems of this study are 1) selection of genes for transfection and 2) efficiency of local gene delivery. The latter problem would be solved by modification or development of vectors for high transfection efficiency and repeated use. The former problem would be solved by identification of genes as this study. However, additional problem will arise where each gene should be transfected.
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上皮由来抗菌ペプチドの利用による歯周病原性細菌の感染予防にむけて
Grant number：11877366 1999 - 2000
日本学術振興会 科学研究費助成事業 萌芽的研究
高柴 正悟, 苔口 進, 前田 博史, 明貝 文夫
Grant amount：\2000000 （ Direct expense: \2000000 ）
ヒト上皮細胞の産生するヒト型ベータデフェンシン-2(HBD-2)には,サイトカインや細菌内毒素の刺激によってその産生が誘導される特徴があるので,感染頻度の高い口腔内において,HBD-2の感染予防治療への応用が期待される。本研究では,HBD-2の発現様態と転写制御因子を解明することを目的とした。
歯周病罹患歯肉中にHBD-2のmRNA発現を検出し,そのcDNAをクローニングした。また,同歯肉を用いた組織免疫染色によって歯肉上皮顆粒層にHBD-2を検出した。この遺伝子をテトラサイクリンによって転写活性を制御することができる哺乳動物発現ベクターpTRE-Mycに挿入し,子宮頚部上皮癌細胞にin vitro遺伝子導入を行って,テトラサイクリンを添加することによってHBD-2遺伝子(hbd-2)の転写を制御した。そして,この細胞の溶解液中のHBD-2の産生をELISA法によって確認した。さらに,この細胞溶解液は大腸菌に対して抗菌活性を示すことがわかった。今後は,ラット等を用いたin vivoにおける遺伝子導入において,テトラサイクリンによる制御を試みる必要がある。一方,導入と発現の効率を向上させるために,海外の研究者の協力の下,ウイルス由来の新規発現ベクターにhbd-2を挿入している途中である。
また,hbd-2プロモーターをクローニングし,β-ガラクトシダーゼをレポーター遺伝子として有しているpSEAPベクターに挿入した。これを子宮頚部上皮癌細胞に導入し,細菌内毒素刺激下での同細胞の産生するβ-ガラクトシダーゼ活性を測定することによって,hbd-2プロモーター領域中の転写制御領域を特定することを試みた。その結果,転写開始点から352bp上流領域に制御部位が存在することがわかった。今後は,この部に結合する転写制御因子を特定する必要がある。 -
Studies on bone regeneration by application of osteoblast differentiation marker, Cbfal, gene products
Grant number：10671967 1998 - 1999
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
MYOKAI Fumio, TAKASHIBA Shogo, MURAYAMA Yoji
Grant amount：\3200000 （ Direct expense: \3200000 ）
For achievement of periodontal tissue regeneration, it is needed to allow the regeneration related factors express in vivo. These factors must act as differentiation and proliferation factors specific for periodontal tissues, have to be elucidated their characters and functions. In this study, we assessed the gene expressions for candidates including Cbfal, and proposed the possibility to introduce gene into periodontal fibroblasts.
Experimental results for the specific points :
1) Gene expression of candidates during rat alveolar bone regeneration
We examined what gene was differentially expressed during alveolar bone healing following artificial defect. Reverse Northern analysis revealed that transcripts for Cbfal, BGP, TGF-β its receptor type III during wound healing. Moreover, unique genes were isolated from periodontal tissues during wound healing.
2) Gene transfer in cell in vitro
Human gingival fibroblasts were co-cultured with IL-1β introduced COS-1 cells. IL-8 mRNA was detected in gingival fibroblasts nearby the COS-1 cells. This result suggests possibility of gene transfer and its application in vivo. -
Study on the pathogenesis of periodontal disease in patients with Down's syndrome
Grant number：10671966 1998 - 1999
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
NISHIMURA Fusanori, TAKASHIBA Shogo, KOKEGUCHI Susumu, EGUSA Masahiko
Grant amount：\3000000 （ Direct expense: \3000000 ）
Patients with Down's Syndrome have been reported to be accompanied by severe periodontal disease. To try to understand the underlying mechanisms behind this, we assessed neutrophil functions in patients with Down's Syndrome. Additionally, patients with Down ; s Syndrome are known to suffer accelerated aging. From the viewpoint of premature aging, we evaluated regenerative capacity of periodontal ligament cells in aged individuals. The results of the study are as follows.
(1) Neutrophil chemotaxis against FMLP,C5a and IL-8 was not impaired in patients with Down's Syndrome when compared with age-matched healthy subjects.
(2) Periodontal ligament cells obtained from aged subjects showed shorter replicative life span as compared with those from juvenile donors.
(3) Periodontal ligament cells from aged subjects showed less chemotactic responses to b-FGF as compared with those from juvenile donors.
(4) Cells reached fully senescence did not express c-fos.
(5) Although migrated cells expressed c-fos , there observed many cells which did not express c-fos in unmigrated cells.
These results suggest that c-fos is necessary for cell migration as well as cell proliferation, and failure to express c-fos may be one of the reasons for low regenerative capacity seen in aged subjects. We conclude that impaired regeneration rather than low neutrophil function may be involved in the high prevalence of periodontal disease seen in Down's Syndrome patients. -
The diversified research on. the genes related periodontal disease
Grant number：10357020 1998 - 1999
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
KURIHARA Hidemi, SUGAI Motoyuki, OKADA Hiroshi, ABIKO Yoshimitsu, YAMAZAKI Kazuhisa, TAKASHIBA Shogo
Grant amount：\25300000 （ Direct expense: \25300000 ）
In this project, we evaluate the genes related periodontal disease from the diversified aspects, infected bacteria, host defense system, tissue metabolism, and tissue regeneration. Analysis of DNA sequence of hemagglutinin from Porphyromonas gingivalis revealed its functional domain. The binding domain contains the specific sequence, PVQNT, similar to influenza virus. A novel gene (Aa cdt) encoding cytolethal distending toxin (CDT) was successfully cloned from Actinobacillus actinomycetemcomitans. Aa cdt was composed with three clusters, and each cluster was encoding CDT-A, -B, and -C. CDT-A, -B and -C were essential for expressing CDT activity.
The genes related autoantibody production in patients with periodontitis were evaluated on T cell receptor (TCR) and human leukocyte antigen (HLA). The peripheral T cells were stimulated by hrHSP60 or P. gingivalis GroEL and DNA sequence of CDR3 region of their TCR β-chain. The amino-acid sequenee of CDR3 was the same between the expanded T cell clone and the accumulated T cell clone in inflamed gingival tissue. The HLA class II genotypes of patients with periodontitis, autoantibody production and PVB 19 infection were analyzed by PCR-RFLP. The frequencies of DQA1 *0101, DQA1 *0501, and DQB1*0503 in patients with periodontitis, autoantibody production and PVB 19 infection were higher than in other patients and healthy subjects. The relationship between IL-1 genotypes and prevalence of adult periodontitis was reported in the United State, however we could not found this relationship in Japanese.
Two patients with hypophosphatasia, usually accompanied with periodontitis, and their family members were analyzed to detect mutation on tissue-nonspecifie alkaline phosphatase gene. Novel three point mutations on the alkaline phosphatase gene were revealed
Novel genes related periodontal tissue regeneration were found by the subtraction method between wound periodontal tissue under tissue repair and healthy periodontal tissue. Sixteen novel cDNAS in enhanced expression genes and 9 novel cDNAS in depressed expression genes were successfully cloned. -
Differentiation from dental pulp cells to odontoblasts and calcification of the pulp.
Grant number：09307045 1997 - 1999
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
UYENO Kazuyuki, TAKASHIBA.SHOGO, KATOH Yoshiroh, IWAKU.KAZUYUKI, IZUMI Toshio, FUJIITANI Morioki
Grant amount：\30900000 （ Direct expense: \30900000 ）
The reconstruction of damaged dentin by activating odontoblasts is important to save teeth for a long time. Various growth factors are thought to have a close relation with the differentiation from pulp cells to odontoblasts and the phenomenon of pulpal calcifications, but those details are not still clarified. The present comprehensive studies using dental pulps of human and animal were performed to clarify the differentiation of pulp cells and pulpal calcifications.
Results are as follows. A system of the organ culture, the possiblity of differentiation, roles of nerve terminal in dentin bridge formation, pulpal calcification in severe periodontal disease, and effects of 1,25-dehydroxyvitamin DィイD23ィエD2 on the expression of osteocalcin by TGF-β and bFGF etc. were shown in human studies. Gene expression and delivery to pulpal cells, effects of bFGF on hard tissue formation in root apex, pulpal response after cavity preparation by Er:YAG laser, dentin-bridge formation by multiple fluorescent labeling, new developments on adhesive resinous materials having a calcification promoting function as direct pulp capping agent, in vitro mineral induction by insoluble dentin collagen, establishment and characterization of a culture system for enzymatically released rat dental pulp cells etc. were shown in animal studies.
This study suggested that various factors made effects on the differentiation of pulp cells and pulpal calcification, and that non-biomaterials in part might have a function as a clinical application. Moreover, some factors about periodontal disease may affect dystrophic calcification of dental pulp. -
Study of Periodontal Tissue Reconstruction using Various Growth Factors
Grant number：09307041 1997 - 1999
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
KATO Hiroshi, KAWANAMI Masamitsu, KAMEYAMA Yoichiro, MAEDA Katsumasa, ODA Shigeru, TAKADA Takashi
Grant amount：\38600000 （ Direct expense: \38600000 ）
(1) Reconstruction of periodontal tissue by application of rhBMP.
Different studies revealed that there are little differences in tissue reactions to partially purified bovine BMP and to rhBMP. To use BMP clinically, the effects of rhBMP on periodontal tissue cells and in animals were investigated using polylactate-polyglycolate gelatine sponge complexes (PGS) as a carrier. There were no adverse or immunological reactions in those experiments. The results showed that rhBMP increases ALP activity of HPLC; regenerates alveolar bone, cementum, and peridontal ligament ; and also induces new bone formation in old rats.
(2) Investigation of periodontal regeneration using PDGF, IGF, TGF-β, and b-FGF.
The effects of PDGF, IGF, TGF-β, and b-FGF on periodontal tissue cells were investigated. Different studies reported that PDGF-BB stimulates HPLC migration and proliferation when applied to the root surface, and prevents ankylosis in the damaged region of the periodontal ligament after tooth replantation. It was showed that IGF and b-FGF increase the proliferation of PLC; TGF-β increases the proliferation of gingival fibroblast, and combined use of these factors may increase the proliferation of periodontal tissue cell.
(3) Investigation of the new growth factors, contained in dentin and periodontal tissue
The growth factors in dentin with TGF-β regulate osteoblast and influence development, remodeling, and regeneration of mineralized tissues. Cementum-derived growth factor (CGF) in cementum increases the proliferation of periodontal ligament cells.
(4) Summary and future research subjects
The findings of this study suggest that rhBMP is useful for thereconstruction of periodontal tissue, and other growth factors have various effects on periodontal tissue for periodontal regeneration. In the future it is necessary to study the useful effects of growth factors singly or in combination for periodontal tissue reconstruction. -
Basic studies on adaptation the immune therapy to the patients with periodontal diseases by using synthetic peptides as an immunogen
Grant number：09470425 1997 - 1998
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
MURAYAMA Yoji, OHYAMA Hideki, NISHIMURA Fusanori, TAKASHIBA Shogo
Grant amount：\12000000 （ Direct expense: \12000000 ）
As the first trial to adapt the immune therapy to the patients with periodontal diseases, we estimated how Ag53 is recognized by host immune cells and derive subsequent effector functions. We determined major T cell epitopes of Ag53 in EOP patients, and estimated them from the viewpoint of the restriction of HLA class II molecules. The results of this study are as follows.
(1)We established Ag53 specific short-termed T cell lines from 22 subjects including 6 EOP patients and 16 healthy donors, using overlapping peptides based on Ag53 amino acid sequences. We found that all T cell lines from active EOP patients recognized common region (pl4l-p18l) on Ag53, while those of healthy donors showed heterogeneous specificity
(2)Anti-DRBL monoclonal antibody inhibited Ag53-induced proliferation of most of the T cell lines.
(3)A large amount of IFN-gamma was produced from all of established Th cell lines. The other cytokine production, including IL-4, 5, 6 and 10, were different among the Th cell lines.
(4)The effect of the cytokine-profile produced from Th cells on the lgG production from B cells was evaluated. The Th cell lines producing high amount of Th2 type cytokines against Thl cytokines induced high IgG production. But the Th cell lines, which produced low Th2 cytokines against Thl cytokines. Did not induce the IgG production.
(5)A larger amount of IL-5 from Th cells was detected in the culture with IgG production. compared to the culture without IgG production.
These results suggest that AA residues from 14 1 to 181 is supposed to include major T cell epitope on Ag53 for active EOP patients but not for healthy individuals or inactive EOP patients. which might be interested in the establishment of the immune therapy for P gingivalisx infection in periodontal diseases -
Studies on differentiation and proliferation factors for induction of biological reparative dentin formation
Grant number：09671951 1997 - 1998
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
TAKASHIBA Shogo, WASHIO Norifumi
Grant amount：\3500000 （ Direct expense: \3500000 ）
For the effective induction of secondary dentin, it is needed to allow the secondary dentin-inducible factors express in dental pulp. These factors must act as differentiation and proliferation factors specific for dental pulp cells, and have to be elucidated their characters and functions, In this study, we succeeded followings : 1) establishment of method to isolate unique genes from tissue and cells (eukaryote and prokaryote), 2) screening of cavity preparation-induced gene in dental pulp. and 3) transfer of stains to pulp chamber through dentinal tubes.
Experimental results for the specific points.
1. Genes expressed uniquely in dental pulp after cavity preparation
To isolate these genes, we needed precise method to detect change of gene expression from small amount of tissue. Polymerase chain reaction (PCR)-based subtractive hybridization (SI-I) method allowed us to isolate cDNAs uniquely expressed in dental pulp after cavity preparation.
2. Model for the transfer of genes or their products to pulp chamber through dentinal tubules
Freshly extracted human teeth which contain vital pulp tissue were used for establishment of this model. The teeth were prepared for organ culture for several days after cavity preparation. Stains were placed into the cavity at the beginning of culture, and found to be in pulp chamber after a couple of days. This result suggests that the possibility of gene transfer to pulp tissue through dentinal tubules.
3. Genes expressed uniquely by human periodontal ligament (PDL) fibroblasts
By subtractive hybridization between PDL fibroblasts and gingival fibroblasts (both are primary culture), several genes were elucidated to be expressed uniquely by PDL fibroblasts. This experiment allowed us to improve SH.
4. Difference of genes between Porphyromonas gingivalis strains
Genes of Porphyromonas gingivalis strains were compared at genomic DNA level by SH.This result was used to apply this method for eukaryotic cells. -
L-セレクチンを介したβ_2-インテグリンのシグナル伝達機構から捉える歯周病病態
Grant number：09671952 1997
日本学術振興会 科学研究費助成事業 基盤研究(C)
葛城 教子, 高柴 正悟
Grant amount：\1700000 （ Direct expense: \1700000 ）
我々は以前に,β_2-インテグリン発現異常のある2名の早期発症型歯周炎(EOP)患者を発見した。この研究結果から,β_2-インテグリンは歯周病を規定しているマーカーとなりうることが示唆された。β_2-インテグリン遺伝子を歯周病の遺伝子診断のマーカーとして確立するためには,歯周病患者におけるβ_2-インテグリンのシグナル伝達機構を解析する必要がある。白血球の組織浸潤過程におけるrollingからstickingへの運動は,L-セレクチンを介してシグナルが伝達され,β_2-インテグリンが活性化されることによって誘導されることが分かっている。我々はまず,上記のEOP患者を含む歯周病患者由来のB細胞株を用いて,β_2-インテグリンの発現をL-セレクチンの発現機能との関わりにおいて調べた。
被験細胞として,13名の被験者(EOP患者9名および健常者4名)の末梢血から樹立したEBウィルストランスフォームB細胞株を用い,以下の結果を得た。
1.無刺激時のL-セレクチンの発現量は,被験細胞株間で差がなかった。β_2-インテグリン発現異常のあるEOP患者を含む4被験細胞では,PMA刺激によるL-セレクチンの発現減少の割合が,健常者を含む他の9被験細胞に比べて少なかった。
2.PMA刺激時の上記の4被験細胞では,B細胞培養上清のsoluble L-セレクチン量が健常者を含む他の9被験細胞に比べて少なかった。
以上の結果から4名のEOP患者由来のB細胞株は,他のEOP患者および健常者のそれらとはL-セレクチンの発現量に違いがあり,L-セレクチンの発現機序が異なっていると考えられる。本研究において,L-セレクチンを介したβ_2-インテグリンのシグナル伝達機構のなかで,L-セレクチンの発現機能が一般的でないEOP患者4名が存在することが分かったので,それらの病態を規定する遺伝子が分子生物学的に調べるなら,“歯周病関連遺伝子"の解明となる。 -
Study on the control of periodontal ligament fibroblast functions by transforming growth factor
Grant number：08457506 1996 - 1998
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
ARAI Hideo, WASHIO Norifumi, MYOKAI Fumio, TAKAHASHI Keiso, NISHIMURA Fusanori, TAKASHIBA Shogo
Grant amount：\4800000 （ Direct expense: \4800000 ）
TGF-beta has been shown to play a critical role in the stimulation and regulation of the wound healing process. Some studies focused on the characteristic of TGF-beta as a biological mediators in periodontal tissue. The purpose of this study is to understand the role of TGF-beta in periodontal tissue regeneration. We examined the effect of TGF-beta on the cellular functions of human periodontal ligament fibroblasts (HPLF) and the kinetics of the expression of TGF-beta and their receptors in HPLF.We got the following results.
1. TGF-beta enhanced DNA synthesis, collagen synthesis, non-collagen synthesis and alkaline phosphatase (ALP) activity as a bone formation marker in HPLF in vitro.
2. HPLF expressed TGF-beta genes and TGF-beta receptor genes (type I, II, III) in vitro
3. The expression of TGF-beta genes and TGF-beta receptor genes decreased in response to assumptive mechanical stress such as a occlusal force in cultured HPLF.In vivo TGF-beta genes were detected in periodontal ligament cells of rat periodontal tissue by using in situ hybridization. The amount of TGF-beta gene expression in periodontal ligament cells is larger than gingival fibroblasts.
These results suggest that TGF-beta plays important role in regulation several key cellular functions of HPLF such as proliferation, cell matrix synthesis, and metabolism of hard tissue in periodontal tissue regeneration and that the mechanisms are regulated in autocrine system and in response to mechanical stress. -
Regulation of osteogenic function of periodontal ligament fibroblast by 1d, 25-dihydroxy vitamin D3 receptor inducing factor.
Grant number：08672187 1996 - 1997
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
TAKIGAWA Masayuki, TAKAHASHI Keiso, NISHIMURA Fusanori, TAKASHIBA Shogo, ARAI Hideo
Grant amount：\2200000 （ Direct expense: \2200000 ）
Regulation of osteogenic function of periodontal ligament fibroblast by 1alpha, 25-dihydroxyvitamin D3 receptor inducing factor
Cellular function of periodontal ligament fibroblasts (PLF) is regulated not only by exogenous factors such as hormones and cytokines, but also by endogenous factors derived from PLF.We have previously demonstrated that multilayred PLF produce a factor (s) which can induce the expression of 1alpha, 25-dihydroxyvitamin D3 receptor (VDR) in an autocrine manner. In this study, we first examined whether known growth factors could also induce the expression of VDR in PLF.The expression of VDR mRNA was induced by IGF-I,-II and EGF,but not by TGF-beta. This indicated that the expression of VDR in PLF could be regulated by several autocrine factors including these growth factors. We, therefore, tried to identify a gene (s) which is specific for PLF,and aimed to study the functions of PLF from a view point of the charactaristics of specific gene. Using confluent PLF and gingival fibroblasts (GF), we constructed a subtraction cDNA library which possibly contained several specific genes for PLF.34 clones were identified by Southern hybridization ; 5 were unknown genes and the other were highly homologous with a part of some known genes. Among the known genes, nm 23 protein or v-fos transformation effector protein, which were related to cell differentiation and proliferation, were included. These specific genes we obtained also might be related to some other important functions of PLF besides cell differentiation or osteogenic function, because they were obtained in comparison with GF from a same individual. It is necessary to classify these genes in the functions of PLF,and to examine whether the expression pattern of the specific gene is different among the differentiation stages of PLF. -
Molecular biological study on tissue regeneration
1996
Grant type：Competitive
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Analyzes of periodontitis status from changes in gingival fibroblasts subpopulation
Grant number：06671909 1994 - 1995
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (C)
ARAI Hideo, WASHIO Norifumi, HONGYO Hiroshi, TAKASHIBA Shogo
Grant amount：\2200000 （ Direct expense: \2200000 ）
Tumor necrosis factor (TNF-alpha) has been known to stimulate prostaglandin production in gingival fibroblasts. In this study, we showed that, by the addition of interleukin-1beta (IL-1beta) into the culture, this enhancement was further augmented. IL-1beta decreased the number of TNF-alpha receptor and affinity to its ligand in gingival fibroblasts. We therefore examined the expression profile of IL-1 receptor mRNA and that of TNF-alpha receptor subtype mRNA under the absence or presence of IL-1beta to understand the underlying mechanisms by which IL-1beta modulate the responsiveness of gingival fibroblasts to TNF-alpha.
Both type I and II TNF-alpha receptor mRNA were detected in gingival fibroblasts, but the expression of type I receptor was much more than that of type II.When gingival fibroblasts was stimulated by IL-1beta, the expression of type I TNF-alpha receptor was markedly depressed, while type II receptor was not affected.
These results indicate that the initial observation that IL-1beta enhances the affinity of TNF-alpha to gingival fibrolasts (leading to increase of PGE_2 synthesis) can not be explained solely by the expression profile of TNF-alpha receptors. -
Molecular biological study on the IL-8 production in inflamed gingiva
Grant number：06671912 1994 - 1995
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (C)
TAKASHIBA Shogo, WASHIO Norifumi, MURAYAMA Yoji
Grant amount：\2100000 （ Direct expense: \2100000 ）
In the initial steps of an immune reaction or inflammation in the skin and mucosa, the following cytokine cascade was postulated to occur in response to inflammatory stimuli such as lipopolysaccharide (LPS) , 1) inflammatory stimuli induced production of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) , 2) proinflammatory cytokine IL-1 and TNF-alpha induced production of leukocyte chemotactic and activating peptide/IL-8. This postulation was examined in mouse skin and in dermal fibroblasts cultured from it. By in situ hybridization, expression of the IL-8 gene was detected in nearly all cultured dermal fibroblasts with or without treatment with human recombinant (hr) IL-1beta and/or hrTNF-alpha. Moreover, cultured dermal fibroblasts were found to express the IL-1beta gene even without treatment. These results suggest that expression of the IL-8 gene in dermal fibroblasts in vitro was induced by proinflammatory cytokines such as IL-1beta presumably functioning in autocrine fashion. However, expression of the IL-8 gene was not induced in dermal fibroblasts in vivo by injecting hrIL-1beta and/or hrTNF-alpha into mouse skin whereas keratiocytes and endothelial cells expressed the IL-8 gene in the skin ; as revealed by in situ hybridization. These results indicate that the process of the IL-8 gene expression elicited by IL-1beta and TNF-alpha in dermal fibroblasts in vitro is quite different from those in keratinocytes and endothelial cells in vivo. Dermal fibroblasts are likely to remain insensitive to IL-1beta and TNF-alpha in non-inflammatory tissues, but become sensitive to them with respect to induction of the IL-8 gene expression in vitro.
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Study on the interleukin-2 producing capacity in the patients with periodontitis
Grant number：04671159 1992.04 - 1993.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (C)
ARAI Hideo, TAKAHASHI Keiso, KOBAYASHI Yoshitomo, KURIHARA Hidemi, MURAYAMA Yoji
Grant amount：\2200000 （ Direct expense: \2200000 ）
The purpose of this study was to assess the role of interleukin-2 (IL-2) in the pathogenesis of periodontitis. The levels of IL-2 produced from mononuclear cells were measured after stimulating with a CD3 monoclonal antibody (CD3 mAb) in 45 patients (12 with juvenile periodontitis [JP], 20 with rapidly progressive periodontitis and 13 with adult periodontitis) and 20 healthy subjects (HS). Six subjects including 3 JP patients demonstrated elevated IL-2 level. When phorbol myristate (PMA) was substituted for monocytes, there existed one JP patient with significantly high IL-2 producing capacity, and two JP patients and one HS with low capacity. To investigate the cause of their unusual IL-2 producing capacities, intracellular signal transduction system in T cells was examined. The levels of IL-2 produced upon stimulation with ionomycin and PMA were proportionate to those produced upon stimulation with CD3 mAb and PMA in the subjects examined except one JP patient ; suggesting that the elevated IL-2 producing capacity of this patient is related to the intracellular calcium ion level after stimulation of CD3 molecules. These results suggest that IL-2 producing capacity is responsible for the pathogenesis of a certain type of early-onset periodontitis, and that intracellular signal transduction system is concerned with the imbalance of IL-2 production in some individuals.
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Study on molecular pathogenesis of inflammation
1992
Grant type：Competitive
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Molecular Basis of Leukocyte Adhesion Molecules in Early-onset Periodontitis Patients.
Grant number：03454441 1991 - 1993
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (B)
MURAYAMA Yoji, SHIMIZU Hideki, TAKASHIBA Shogo, TAKAHASHI Keiso, ISOSHIMA Osamu, KURIHARA Hidemi
Grant amount：\6500000 （ Direct expense: \6500000 ）
We analyzed the cell-cell adherence related to CD11/CD18 and CD18 mRNA of the individuals with decreased CD11/CD18 expression on the surface of their neutrophils. Epstein Barr virus-transformed B cell lines were developed from one localized juvenile periodontitis (LIP) patient characterized by decreased CD11/CD18 on the neutrophils in peripheral blood and without any remarkable systemic diseases, two siblings with generalized prepubertal periodontitis (GPP) caused from leukocyte adhesion deficiency (LAD), another LIP patient, one localized prepubertal periodontitis (LPP) patient, and two healthy subjects. Adhesion of leukocytes to each other was measured as cluster formation by aggregation assay. The length and the amount of CD18 mRNA expressed in the cell lines were analyzed by Northern blotting using the 32p-labeled CD18 cDNA.The coding region of the mRNA was analyzed by the reverse transcription-polymerase chain reaction method. Base-mismatches between CD18 mRNA and the 32p-labeled RNA probe synthesized from Cd18 cDNA were analyzed by RNase protection assay. In the adherence assay, cells from the LIP patients with decreased CD11/CD18 formed more clusters of smaller size and with fewer cells than those of did the patients and healthy subjects, while the cells from both GPP patients were found not to aggregate and not to form clusters either in the absence or presence of PMA.There were no differences in the length and the amount of mRNA between the LIP patient and the other patients and healthy subjects, while GPP patients expressed a small amount of long mRNA.The whole coding region (2,313 base pairs) was amplified from all of subjects except the GPP Patients, while the 5'-region (1,119 base pairs) was amplified from all subjects. Base-mismatches were detected on the coding region from 965 to 1,450 nucleotides of CD18 mRNA in GPPpatients. No mismatch was detected on other regions of CD18 mRNA in any subject. These results suggest that the LIP patient with an anom
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歯周病患者に検出されたHLA遺伝子変異の解析
Grant number：03857257 1991
日本学術振興会 科学研究費助成事業 奨励研究(A)
高柴 正悟
Grant amount：\900000 （ Direct expense: \900000 ）
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Study on genetics of periodontal diseases
Grant type：Competitive