Research Projects -
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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.