Studies on the Mechanism of Calcification - On a Protease Associated with Matrix Vesicles -

钙化机制的研究 - 与基质囊泡相关的蛋白酶 -

• 批准号: 61440082
• 负责人: ASAAKI Kawamura
• 金额: $ 13.31万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 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/grant/KAKENHI-PROJECT-61440082/
• 关键词: Calcification; Protease of Matrix Vesicles; Culture System; Calcifying Matrices; コラーゲン; 石灰化; 基質小胞; タンパク分解酵素; プロテオグリカン

In this study, in order to analyse the proteases of matrix vesicles in calcification, we aimed to establish the culture system in which the process just like in vivo calcification was expressed.For its purpose, we used the established osteogenic cells, MC3T3-El which possessed high collagen synthesis activity, matrix vesicle-producing activity and calcification activity.Primarily we examined definite culture condition and compatibility of FCS to cells. Secondarily we confirmed that calcified tissues produced by El cells were composed of hydroxyapatite by electron diffraction analysis and energy disperse x-ray analysis. Finally we analysed biochemical changes in this culture system. Consequently we observed the increase of AL-P activity, the decrease of hexosamines and the synthesis of type 1 collagen. It was markedly important that crosslining of collagen in this culture was indicated as same as in vivo and El cells produced matrix vesicle-like substances. It was suggested to be of capability to analyse proteases of matrix vesicle enzymologically in this culture system.Now we are planning to establish the culture system of chondrocytes in epiphyseal cartilage and investigate in detail the qualitative changes of collagen, interaction of noncollagenous components including proteoglycan with it, and significance of proteases of matrix versicles in calcification.

本研究利用已建立的具有高胶原合成活性的成骨细胞MC 3 T3-E1，首先考察了FCS与细胞的相容性及培养条件。其次，通过电子衍射分析和能量色散X射线分析证实了E1细胞产生的钙化组织由羟基磷灰石组成。最后分析了该培养体系中的生化变化。因此，我们观察到AL-P活性的增加，己糖胺的减少和1型胶原的合成。这是显着重要的是，在这种文化中的胶原交联表示在体内和El细胞产生基质囊泡样物质相同。本实验拟建立骺软骨细胞培养体系，详细研究骺软骨细胞胶原质的变化、蛋白多糖等非胶原成分与胶原质的相互作用以及基质囊泡蛋白酶在钙化中的意义。