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延边牛骨骼肌卫星细胞的分离培养及诱导分化

Isolation, culture and induction differentiation of Yanbian cattle skeletal muscle satellite cells

  • 摘要: 为建立体外研究延边牛肌肉分化及成脂相关调控过程的牛骨骼肌卫星细胞原代细胞模型,试验以12日龄延边牛犊牛背最长肌为材料,利用链霉蛋白酶消化和差速贴壁法对延边牛骨骼肌卫星细胞进行分离纯化,期间观察细胞形态变化;利用免疫荧光染色法对特异性标记基因配对盒基因7(Pax7)、结蛋白(Desmin)和成肌分化抗原(MyoD)进行检测鉴定分离细胞;绘制细胞生长曲线;细胞进行成肌诱导分化,利用荧光定量PCR鉴定Pax7和MyoD基因在诱导前后表达量的变化。结果表明:刚分离的骨骼肌卫星细胞贴壁生长后,细胞由圆形向梭形或纺锤形变化,随着培养时间的延长,细胞开始有序生长;骨骼肌卫星细胞特异性标记基因Pax7、Desmin、MyoD免疫荧光染色均呈阳性表达;骨骼肌卫星细胞符合正常的生长规律;细胞成肌诱导分化后,MyoD、Pax7基因在诱导成肌的各阶段均有表达,表明诱导后骨骼肌卫星细胞处于活化状态并向成肌细胞分化。说明本研究成功分离获得了延边牛骨骼肌卫星细胞,可为研究延边牛肌肉分化及成脂相关调控过程提供体外细胞模型。

     

    Abstract: In order to establish a bovine skeletal muscle satellite cells model for studying Yanbian cattle muscle differentiation and adipogenesis-related regulation processes in vitro, the longissimus dorsi muscle of 12-day-old Yanbian calf was selected as the experimental material. The bovine skeletal muscle satellite cells of Yanbian cattle were isolated and purified by streptomysin digestion and differential adherent method, and the morphological changes of the cells were observed. Immunofluorescence staining method was used to detect paired box 7 gene(Pax7), Desmin gene and myoblast differentiation antigen gene(MyoD) to identify the isolated cells. Cell growth curve were plotted. The cells were differentiated by myoblast induction, and the expression levels of Pax7 and MyoD genes before and after induction were determined by fluorescence quantitative PCR. The results showed that after the newly isolated skeletal muscle satellite cells adhered to the wall, the cells changed from round to spindle or spindle shape. With the extension of the culture time, the cells began to grow in an orderly manner. Immunofluorescence staining showed that the specific marker genes(Pax7, Desmin and MyoD) of skeletal muscle satellite cells were all positively expressed. Skeletal muscle satellite cells were consistent with the normal growth law. MyoD and Pax7 genes were expressed at all stages of myogenesis after induction, indicating that the muscle satellite cells differentiated into myoblast cells in the activated state after induction. The results indicated that the satellite cells of Yanbian bovine skeletal muscle were successfully isolated and obtained in this study, which could provide a cell model in vitro for studying muscle differentiation and adipogenesis related regulation process of Yanbian cattle.

     

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