大鼠骨骼肌卫星细胞的原代培养鉴定和体外分化特点

Study on culture, identification and differentiation of primary rat skeletal muscle satellite cells

目的建立骨骼肌卫星细胞（SMSC）的体外分离、原代培养、鉴定方法，观察细胞的成肌分化特点。方法采用组织块法结合差速贴壁法获得SMSC。采用免疫荧光和流式细胞仪以Pax7为标志物鉴定分离获得的卫星细胞纯度，用分化培养基诱导SMSC的体外分化，实时定量PCR法检测分化标志基因成肌决定因子（MyoD）和生肌素的mRNA相对表达量。结果组织块法培养约1周，可见细胞从组织块边缘爬出。经差速贴壁法纯化后，流式细胞仪检测所获得的原代SMSC纯度可达97．6％。细胞体外诱导分化后，MyoD和生肌素基因呈时序性表达。结论组织块法可成功获得高纯度的SMSC，在体外具有良好的分化能力。

Objective To establish the isolation, culture and identification methods of primary rat skeletal muscle satellite cells （SMSC） and observe its characterization of differentiation in vitro. Methods Skeletal muscle satellite cells were obtained by tissue block culture method in combination with pre-plating techniques, and the purity of these cells was detected by both immunocytochemistry and fluorescence activated cell sorter （FACS） with Pax7 as marker of SMSC. Myogenesis of these cells was induced in differentiation medium and the mRNA expressions of myogenic differentiation gene （MyoD） and Myogenin were determined by Real-time polymerase chain reaction （PCR）. Results Cells crawled out from the edge of tissue blocks after 1 week of culture. After purification by pre-plating techniques, more than 97. 6% of the cells expressed PaxT, a unique marker of satellite cells. The mRNA of MyoD and Myogenin showed time- specific expression in the myogenesis induction process in vitro. Conclusion Skeletal muscle satellite cells with high purity and strong differentiation ability can be obtained by means of tissue block culture method.