绵羊骨骼肌卫星细胞分离培养、鉴定与成肌诱导分化

doi:10.16431/j.cnki.1671-7236.2021.04.007

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (4): 1204-1210.doi: 10.16431/j.cnki.1671-7236.2021.04.007

绵羊骨骼肌卫星细胞分离培养、鉴定与成肌诱导分化

李欣¹, 于永生¹, 张立春¹, 马惠海², 罗晓彤³, 魏天², 肖成¹, 张琪¹, 曹阳¹, 赵中利²

1. 吉林省农业科学院动物生物技术研究所, 公主岭 136100;
2. 吉林省农业科学院畜牧兽医研究所, 公主岭 136100;
3. 延边大学农学院, 延吉 133002

收稿日期:2020-08-14 出版日期:2021-04-20 发布日期:2021-04-16
通讯作者: 曹阳, 赵中利 E-mail:caoyang2@163.com;zhaozhongli954@sohu.com
作者简介:李欣(1982-),女,黑龙江牡丹江人,硕士,助理研究员,研究方向:动物干细胞,E-mail:lixin-hd@163.com
基金资助:
吉林省科技攻关项目（20190301006NY）；吉林省地方科技创新引导与扶贫项目（20200702016NC）；国家肉羊产业技术体系（cars38）

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

LI Xin¹, YU Yongsheng¹, ZHANG Lichun¹, MA Huihai², LUO Xiaotong³, WEI Tian², XIAO Cheng¹, ZHANG Qi¹, CAO Yang¹, ZHAO Zhongli²

1. Institute of Animal Biotechnology, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China;
2. Institute of Animal and Veterinary Medicine, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China;
3. Agricultural College, Yanbian University, Yanji 133002, China

Received:2020-08-14 Online:2021-04-20 Published:2021-04-16

摘要/Abstract

摘要： 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。

关键词: 绵羊; 骨骼肌卫星细胞; 分离培养; 免疫荧光鉴定; 成肌诱导分化

Abstract: 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 collagenase Ⅳ and 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.

Key words: sheep; skeletal muscle satellite cells; isolation and culture; immunofluorescence identification; myogenic differentiation

中图分类号:

S826

李欣, 于永生, 张立春, 马惠海, 罗晓彤, 魏天, 肖成, 张琪, 曹阳, 赵中利. 绵羊骨骼肌卫星细胞分离培养、鉴定与成肌诱导分化[J]. 中国畜牧兽医, 2021, 48(4): 1204-1210.

LI Xin, YU Yongsheng, ZHANG Lichun, MA Huihai, LUO Xiaotong, WEI Tian, XIAO Cheng, ZHANG Qi, CAO Yang, ZHAO Zhongli. Isolation,Culture,Identification and Myogenic Differentiation of Sheep Skeletal Muscle Satellite Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1204-1210.

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绵羊骨骼肌卫星细胞分离培养、鉴定与成肌诱导分化

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

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