鸽骨骼肌卫星细胞的分离、培养及成肌特性

【目的】探讨白羽王鸽Columba livia骨骼肌卫星细胞的分离、培养和鉴定的方法,建立完整的家鸽骨骼肌卫星细胞的培养体系。【方法】选择孵化16 d的鸽胚作为试验材料,采用组织块贴壁法和胶原酶消化法分离胸肌的骨骼肌卫星细胞并绘制其生长曲线。待卫星细胞分化出肌管后,采用免疫荧光法检测肌球蛋白重链(MyHC)的表达;在细胞分化出肌管前、后分别提取总RNA,采用RT-qPCR方法检测Desmin、Pax7、MyoG和MyoD1基因在细胞分化出肌管前、后的相对表达量。【结果】组织块贴壁法和胶原酶法均能成功地分离出骨骼肌卫星细胞,其生长曲线呈"S"型;该细胞经含体积分数为20%FBS的DMEM高糖培养液培养7 d后视野中出现大量明显可见的肌管,成肌特异性标志MyHC表达呈阳性。RT-qPCR结果表明,Desmin和MyoG基因分化后的相对表达量分别是分化前的5.68和10.38倍,而Pax7和MyoD1基因分化前的相对表达量分别是分化后的7.01和5.51倍。【结论】建立了鸽骨骼肌卫星细胞的培养体系,为今后进行家鸽肌肉发育的研究提供细胞模型。

Isolation, identification and biological characteristics of skeletal muscle satellite cells in pigeons

Objective To investigate the method of isolation, culture and identification of white king pigeon(Columba livia) skeletal muscle satellite cells, and establish a complete culture system of pigeon skeletal muscle satellite cells.Method We selected 16-day pigeon embryos as experimental materials. Skeletal muscle satellite cells were isolated from the pectoral muscle by the tissue explants adherent method and collagenase digestion method, and the growth curve was drawn. MyHC expression was detected by immunofluorescence assay after differentiation of the satellite cells into myotubes. We detected mRNA expression of Desmin, Pax7, MyoG and MyoD1 genes before and after cell differentiation into myotubes using RT-qPCR.Result Skeletal muscle satellite cells were successfully isolated by the tissue explants adherent method and collagenase method, and cell growth appeared in S-shaped curve. After seven days of culture in high glucose DMEM containing 20% fetal bovine serum(FBS), a large number of visible myotubes were observed, and the myogenic differentiation marker MyHC was expressed in differentiated cells. RT-qPCR results showed that the relative expressions of Desmin and MyoG genes in differentiated myotube cells were 5.68 and 10.38 times of those in skeletal muscle satellite cells before differentiation, while the relative expressions of Pax7 and MyoD1 genes in skeletal muscle satellite cells before differentiation were 7.01 and 5.51 times of those in differentiated myotube cells, respectively.Conclusion The culture method of pigeon skeletal muscle satellite cells has been established,which provides a cell model for future studies of muscle development in pigeons.