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

以往的研究表明，牙龈拟杆菌产生的胶原分解酶被普遍认为是牙周病的病原体之一。这种酶能被土壤细菌产生的物质强烈抑制。本研究通过人牙龈成纤维细胞培养系统的实验，明确该酶抑制剂的特性，探讨该材料是否可用于牙周病的预防，并通过形态学和生物学特征鉴定，确定该酶抑制剂的分离源为土壤细菌。通过微滤膜超滤、Sephadex G-15凝胶过滤、反相高压液相色谱等方法分离和纯化了该菌株。通过这些步骤，最终获得了四个具有胶原溶解酶抑制活性的组分，并进行了氨基酸分析。这些抑制剂是低分子量肽，仅由一至两个氨基酸组成，包括未确定的氨基酸。该底物还抑制从B分离的蛋白酶。而对牛胰胰蛋白酶活性无影响。抑制剂对B来源的胶原溶解酶的致细胞病变有较强的预防作用。gingivalis已经生长至汇合的人牙龈成纤维细胞（HGF）在与胶原溶解酶进一步孵育时受到严重损伤。然而，本研究分离的抑制剂的存在防止了HGF细胞的形态学损伤。提示从链霉菌中分离出的胶原酶抑制剂能维持牙龈细胞的正常更新，预防牙周病的发生。

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.