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

以往的研究表明，牙龈拟杆菌产生胶原溶解酶，被普遍认为是穿孔牙病原体之一。这种酶被一种土壤细菌产生的物质强烈抑制。本研究的目的是阐明该酶抑制剂的特性，并通过人牙龈成纤维细胞培养系统的实验，探索该材料是否可以应用于牙周病的预防。作为胶原酶抑制剂分离源的土壤细菌根据其形态和生物学特性鉴定为链霉菌之一。通过微型模组膜超滤、Sephadex G-15凝胶过滤、反相高压液相色谱等方法分离纯化该抑制剂。通过这些步骤，最终获得了作为胶原酶抑制剂的四个活性组分，并进行了氨基酸分析。抑制剂是低分子量肽，仅由一到两个氨基酸组成，包括未确定的氨基酸。该物质还能抑制从牙龈芽孢杆菌中分离的蛋白酶，但对牛胰腺胰蛋白酶的活性没有抑制作用。这些抑制剂对牙龈芽孢杆菌胶原溶酶的细胞致病性有较强的预防作用，人牙龈成纤维细胞（HGF）已生长到汇流状态，再与胶原溶酶孵育后损伤严重。然而，本研究中分离的抑制因子g的存在阻止了HGF细胞的形态损伤。这些结果表明，从链霉菌中分离的这种胶原溶解酶抑制剂可以维持牙龈细胞的正常周转，防止牙周病的发生。

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.