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ANALYSIS OF SECRETORY PROTEINASES AND THEIR INHIBITORS INVOLED IN MYOBLAST DIFFERENTIATION USING SERUM-FREE CULTURE SYSTEM

无血清培养系统分析成肌细胞分化中的分泌蛋白酶及其抑制剂

基本信息

批准号：
11680705
负责人：
YASUMITSU Hidetaro
金额：
$ 2.11万
依托单位：
Yokohama City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

For the detailed investigation of extracellular signaling mechanism on the mammalian myoblasts differentiation, in vitro serum-free culture system was needed. To establish it, several kinds of media and supplements were tested and their concentrations were compared. As the result, we established serum-free culture conditions for myoblasts differentiation and the system provides reproducible results and induces myogenesis more efficiently than the previously reported ones with low serum concentration.To detect and characterize extracellular matrix (ECM) processing proteinases, we tried to improve ''zymography". Serine proteinases and metalloproteinases are known to be involved in the ECM processing. To optimize the conditions for two kinds of enzymes, buffer pII, components and their concentrations were examined. As the results, under the improved conditions metalloproteinases and serine proteinases were detected more sensitive by five-fold and two-fold, respectively, than the previously reported ones.Using improved culture conditions and detection methods, secretory proteinases were detected to vary during myoblasts differentiation. By the addition of proteinous inhibitors for serine proteinases and metalloproteinases, myogenesis were blocked. It demonstrated that the secretory proteinases may play critical roles in myogenesis and may affect in an autocrine manner. Previous reports demonstrated membrane type metalloproteinases family, ADAM play important roles in myogenesis. However, in our system in the presence,of synthetic inhibitor for meltrin, myogenesis was not blocked and it revealed that the contribution of ADAM may not play as a primary actors in the differentiation process.

为了深入研究细胞外信号对哺乳动物成肌细胞分化的影响，需要体外无血清培养体系。为了建立它，测试了几种培养基和补充剂并比较了它们的浓度。因此，我们建立了成肌细胞分化的无血清培养条件，该系统提供了可重复的结果，并且比以前报道的低血清浓度更有效地诱导成肌细胞。为了检测和表征细胞外基质（ECM）加工蛋白酶，我们试图改进“酶谱法”。已知丝氨酸蛋白酶和金属蛋白酶参与ECM加工。为了优化两种酶的条件，缓冲液pII，组分和它们的浓度进行了检查。结果表明，在改进的培养条件下，金属蛋白酶和丝氨酸蛋白酶的检测灵敏度分别提高了5倍和2倍。通过添加丝氨酸蛋白酶和金属蛋白酶的蛋白质抑制剂，肌生成被阻断。这表明分泌性蛋白酶可能在肌肉发生中起关键作用，并可能以自分泌方式起作用。以往的研究表明膜型金属蛋白酶家族ADAM在肌形成过程中起重要作用。然而，在我们的系统中，在meltrin的合成抑制剂的存在下，肌生成没有被阻断，并且它揭示了ADAM的贡献可能不是分化过程中的主要参与者。