绵羊骨骼肌卫星细胞分离培养、鉴定与成肌诱导分化

试验旨在分离绵羊骨骼肌卫星细胞（skeletal muscle satellite cells,SMSCs）,建立绵羊SMSCs体外分离、培养及鉴定体系,为后续研究提供种子细胞。以新生健康绵羊为试验动物,采用胶原酶Ⅳ和胰酶两步酶消化法和差速贴壁法分离并纯化SMSCs。用RT-PCR和免疫荧光法鉴定SMSCs标记基因配对盒基因7（paired box 7,Pax7）、结蛋白（Desmin）和生肌调节因子1（myogenic regulatory factors 1,MyoD1）的表达情况;用血清撤离法诱导SMSCs向成肌细胞方向分化,成肌诱导后观察肌管的形成,免疫荧光法检测成肌分化特异性标志肌球蛋白重链（myosin heavy chain,MHC）的表达。RT-PCR结果显示,扩增条带与预期相符,所分离细胞表达SMSCs标记基因Pax7、Desmin和MyoD1;免疫荧光鉴定结果显示,所分离细胞表达SMSCs标记蛋白Pax7、Desmin和MyoD1;成肌诱导后镜下可见细胞相互融合形成多核的肌管,并表达成肌特异性标志MHC。本试验分离了绵羊SMSCs,建立了适用于绵羊SMSCs的体外培养体系,并成功进行了成肌诱导分化,为今后研究绵羊骨骼肌生长发育机制提供了试验材料和技术支撑。

Isolation,Culture,Identification and Myogenic Differentiation of Sheep Skeletal Muscle Satellite Cells

This experiment was aimed to isolate sheep skeletal muscle satellite cells(SMSCs),establish an optimal system for isolation,culture and identification of sheep SMSCs in vitro,thereby laying a seed cells for future pertinent researches.The SMSCs were isolated from new-born healthy sheep,disassociated with collagenaseⅣand trypsin and purified via differential adhesion method.RT-PCR and immunofluorescence methods were used to identify the expression of SMSCs marker genes paired box 7(Pax7),Desmin and myogenic regulatory factors 1(MyoD1),and serum withdrawal method was used to induce SMSCs to differentiate into myoblasts.Satellite cells underwent muscle differentiation and formed myotubes.Muscle differentiation marker myosin heavy chain(MHC)in cytoplasm were detected by immunofluorescence.RT-PCR results showed that the amplified bands were consistent with the expected results,indicating that the cells expressed SMSCs marker genes Pax7,Desmin and MyoD1.The cultured cells expressed marker proteins Pax7,Desmin and MyoD1,demonstrating that SMSCs were obtained.Muscle differentiation marker MHC was detected in these cells.From the above results,the isolated and cultured cells were SMSCs,and a suitable in vitro culture system for sheep SMSCs was established,and myogenic differentiation was successfully carried out,which provided experimental materials and technical support for the future research on the mechanism of sheep skeletal muscle growth and development.