Research Projects -
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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。