Research Projects -
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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