猪骨骼肌卫星细胞体外分离培养研究进展

猪骨骼肌是动物机体重要的运动组织及人类主要的肉食来源,也是研究肌肉生长发育和疾病的良好模型。猪出生后,骨骼肌的生长发育、损伤修复都需要肌卫星细胞的参与,体外分离培养猪骨骼肌卫星细胞是深入研究骨骼肌生长发育及疾病发生机理的基础,是在细胞水平进行分子功能验证的前提。随着肌肉发育和病理分子机制研究的不断深入,猪骨骼肌卫星细胞的体外分离培养技术也迅速发展起来。背最长肌、后腿肌和半腱肌常用于分离骨骼肌卫星细胞,1日龄猪背最长肌的分离效果最好。常用于分离骨骼肌卫星细胞的酶包括链酶蛋白酶、胶原酶、胰蛋白酶、胶原蛋白酶等,各酶及酶联合消化的时间不同,最优的过滤方式是200目+400目联合过滤,3次离心法可获得纯度较高的细胞。常使用的培养基为DMEM/F12+10%胎牛血清(FBS)+1%青-链霉素(P/S)。骨骼肌肌卫星细胞常见标记物有配对盒基因3(PAX3)、PAX7、生肌决定因子5(Myf5)、Myf4、肌分化因子(MyoD)、肌细胞生成素(MyoG)等。作者通过对猪骨骼肌肌卫星细胞的分离、培养及鉴定等方面进行综述,梳理出各步骤中最佳参数,为建立规范猪骨骼肌卫星细胞分离程序提供参考,以期为肌肉发育和疾病研究提供理论及技术支持。

Research Progress on Isolation and Culture of Porcine Skeletal Muscle Satellite Cells in vitro

Porcine skeletal muscle is an important movement organization of animal body,the main meat source of human beings,and a good model for muscle growth and disease research.After birth,pig needs the participation of muscle satellite cells in the growth and development of skeletal muscle,and the damage repair.Isolation and culture of porcine skeletal muscle satellite cells in vitro is the basis for in-depth study on the growth and development of skeletal muscle and the pathogenesis of diseases,and the premise for molecular function verification at the cellular level.With the deepening of the research on muscle development and pathological molecular mechanism,the technology of isolation and culture of satellite cells of porcine skeletal muscle in vitro has developed rapidly.The longissimus dorsi muscle,hind leg muscle and semitendinosus muscle are commonly used to isolate skeletal muscle satellite cells,and the longissimus dorsi muscle of 1-day-old piglets have the best isolation effect.Enzymes commonly used to isolate skeletal muscle satellite cells include pronase,collagenase,trypsin,collagenase,etc.,and the time of digestion of each enzyme and its combination is different.The best filtration method is 200 mesh +400 mesh combined filtration,and high purity cells can be obtained by three times centrifugation.The usual medium is DMEM/F12+10% fetal bovine serum (FBS)+1% penicillin-streptomycin (P/S).Common markers of skeletal muscle satellite cells include pairing box gene 3(PAX3),PAX7,Myo-genic determinant 5(Myf5),Myf4,Myo-differentiation factor (MyoD),myogenin (MyoG) and so on.In this paper,the isolation,culture and identification of muscle satellite cells of porcine skeletal muscle were reviewed,and the best parameters in each step were sorted out,which provided references for establishing a standardized procedure for isolating porcine skeletal muscle satellite cells,in order to provide theoretical and technical support for muscle development and disease research.