Research Projects -
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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