牛骨骼肌卫星细胞的分离培养及诱导分化方法的建立

为了给牛骨骼肌卫星细胞的分离培养及诱导分化方法及进一步揭示肌肉分化的机理及转基因肉牛的研究提供重要帮助,试验以新生胎牛的骨骼肌为试验材料,分别采用胶原酶Ⅰ和胶原酶Ⅺ与胰蛋白酶结合对其进行消化,并通过差速贴壁法对骨骼肌卫星细胞进行分离纯化,同时采用免疫荧光染色、RT-PCR、Western-blot法对骨骼肌卫星细胞进行鉴定。结果表明:研究成功获得了大量的牛骨骼肌卫星细胞;该细胞的标志性分子的mRNA及其蛋白表达的纯度在98%以上;细胞生长状态良好,可稳定传至90代并保持旺盛的增殖活力;细胞分化效率高,2%的马血清能够诱导细胞分化使其融合形成多核肌管,数量众多的肌管可自发融合为更粗的肌管,并可观察到其具有收缩现象。

Establishment of the methods for isolation,culture and differentiation induction of satellite cells from bovine skeletal muscle

To further reveal the mechanisms of muscle differentiation and provide important help for transgenic cattle research.The skeletal muscles obtained from new-born fetal bovine were used as materials.Collagenase Ⅰ and Ⅺ were combined with trypsin for the digestion,respectively.The skeletal muscle satellite cells were isolated and purified by the differential adhesion method,and then they were also identified using immunofluorescence staining,RT-PCR and Western-blot.The results showed that a large number of bovine skeletal muscle satellite cells were obtained successfully.The purity for mRNA and protein expression of the cell markers was more than 98%.The cells grew in good condition and could passage 90 generations to maintain strong proliferative activity.The cells kept high differentiation efficiency,the cells were fused to form polynuclear myotubes using 2% horse serum to induce the cellular differentiation.Many myotubes could fuse to thicker myotubes spontaneously,and the shrinkage could be observed.