Isolation of collagenase inhibitor from soil bacterium and its application on periodontal disease.

土壤细菌胶原酶抑制剂的分离及其在牙周病治疗中的应用

• 批准号: 61870077
• 负责人: NAKAMURA Ryo
• 金额: $ 3.07万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61870077/
• 关键词: Bacteroides gingivalis; Collagenolytic enzyme inhibitor; Periodontaol disease; Soil bacterium; コラゲナーゼ・インヒビター

pervious study ha shown that Bacteroides gingivalid produces collagenolytic enzyme which is qenerally believed one of perfiodontopathogens. This enzyme is strongly inhibited by the substance(s) prosuced by a strain of soil bacterium. The purpose of this study was to clatilify the characteristics of this enzyme inhibitor and to search, by the experiment using human gingival fibroblast cell culture system, whether this material can be applied for the prevention of periodontal disease.The soil bacterium used for the isolation source of the collagenolytic enzyme inhibitor was identified as one of streptomyces by its morphological and biological characteristics. The inhibitow ras isolated and purified by procedures including an ultrafiitration using minimodule membrane, gel filtration on Sephadex G-15, followed by reverse-phase high pressure liwquid chromatography. Through these procedures, four active fractions as the collagenolytic enzyme inhibitors were finally obrained and subjected to amino acis analyss. The inhibitors were of low molecular weight peptides and consisted of only one to two amino acids including undetermined ones. This substrance also inhibited protease(s) isolated from B. gingivalis, but did not the activity of trypsin from bovine pancreas. The inhibitors had the strong preventive effect on the cytopathogenecity of the collagenolytic enzyme from B. gingivalis Human gingival fibroblast (HGF) cells, which had been grown to confluence, were seberely damaged by the further incubation with the collagenolytic enzyme. However, the presence of the inhibitor isolated gfor this study prevented from morphological damages of HGF cells. These results suggest that this collagenolytic enzyme inhibitor isolated from Streptomyces maintains normal turmnover of gingival cells ans preventsfrom the developemtn of periodontal disease.

以往的研究表明，牙龈类杆菌能产生胶原酶，通常被认为是牙周病的病原体之一。该酶被一株土壤细菌产生的物质(S)强烈抑制。本研究的目的是对这种酶抑制剂的性质进行分类，并通过人牙龈成纤维细胞培养系统的实验，寻找这种材料是否可以用于预防牙周病。通过形态和生物学特性鉴定，作为胶原酶抑制剂分离来源的土壤细菌是链霉菌之一。采用微模膜超滤、Sephadex G-15凝胶过滤、反相高压液层析等步骤分离纯化抑制因子ras。通过这些步骤，最终得到了四个有效的胶原酶抑制剂组分，并进行了氨基酸酶分析。这些抑制剂是小分子多肽，只由一到两个氨基酸组成，其中包括未确定的氨基酸。该底物对牙龈假单胞菌分离的S蛋白酶也有抑制作用，但对牛胰腺胰蛋白酶的活性无明显影响。该抑制物对牙龈假单胞菌胶原酶的细胞病变有较强的预防作用，培养至融合状态的人牙龈成纤维细胞(HGF)在进一步与胶原酶共同孵育后发生严重损伤。然而，本研究中分离的GFOR抑制剂的存在防止了HGF细胞的形态损伤。这些结果表明，这种从链霉菌中分离出来的胶原酶抑制剂可以维持牙周细胞的正常翻转，预防牙周病的发生。

项目成果

Y. Tomobe, J. Endo, D. Hinode, R. Maechara, A. Nagata, J. Shimada, M. Sato and R. Nakamura: "Putification of Thiol-Dependet Collagenolytic Enzyme Isolated from Culture Supernatant of Bacteroides gingivalis 381 and Its Inhibition by Some Serum Componets."

Y. Tomobe、J. Endo、D. Hinode、R. Maechara、A. Nagata、J. Shimada、M. Sato 和 R. Nakamura：“从牙龈拟杆菌 381 及其培养物上清液中分离出的硫醇依赖性胶原蛋白分解酶的腐化”

中村亮,佐藤誠,前原玲子,遠藤順子,日野出大輔,永田篤司,嶋田順子,田中敏之: 日腔衛生学会雑誌.

Ryo Nakamura、Makoto Sato、Reiko Maehara、Junko Endo、Daisuke Hinode、Atsushi Nagata、Junko Shimada、Toshiyuki Tanaka：日本口腔卫生学会杂志。

中村 亮,佐藤 誠,前原玲子,遠藤順子,日野出大輔,嶋田順子: 口腔衛生学会雑誌. 38. (1988)

Ryo Nakamura、Makoto Sato、Reiko Maehara、Junko Endo、Daisuke Hinode、Junko Shimada：口腔卫生学会杂志 38。（1988）。

Y.Tomobe;J.Endo;D.Hinode;R.Maehara;A.Nagata;J.Shimada;M.Saro and R.Nakamura: Jornal Dental Health. 37. 630-636 (1987)

Y.Tomobe；J.Endo；D.Hinode；R.Maehara；A.Nagata；J.Shimada；M.Saro 和 R.Nakamura：《牙科健康杂志》。

Rn. Nakamura, M. Sato, Rn. Maehara, J. Endo. D. Himode, and J. Shimada: "Isolation and Purification of Collagenolytic Enzime Inhibitor from Soil Bacterium and Its Effect on Human Gingival Fibroblast Cells." Journal of Dental Health. 38. (1988)

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