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

为了详细研究哺乳动物成肌细胞分化的细胞外信号机制，需要建立体外无血清培养体系。为了建立它，测试了几种介质和补充剂，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性的结果，并且比以前报道的低血清浓度的系统更有效地诱导肌肉生成。为了检测和鉴定细胞外基质(ECM)加工蛋白酶，我们试图改进‘酶谱’。已知丝氨酸蛋白酶和金属蛋白酶参与细胞外基质的加工。为了优化两种酶的反应条件，对缓冲液PII的组成及其浓度进行了考察。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。利用改进的培养条件和检测方法，检测了成肌细胞分化过程中分泌型蛋白酶的变化。通过添加丝氨酸蛋白酶和金属蛋白酶的蛋白抑制剂，肌肉发生被阻断。提示分泌型蛋白水解酶可能在肌细胞发生中起关键作用，并可能以自分泌的方式发挥作用。以往的报道表明，膜型金属蛋白酶家族，ADAM在肌肉发生中起着重要作用。然而，在我们的系统中，在Meltrin合成抑制剂的存在下，肌肉发生没有被阻止，这表明ADAM的贡献可能不是分化过程中的主要参与者。

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.