北京油鸡骨骼肌卫星细胞的分离、培养、鉴定及成肌诱导分化的研究

【目的】探讨体外培养条件下北京油鸡骨骼肌卫星细胞分离、培养及鉴定的方法,建立适于北京油鸡骨骼肌卫星细胞体外扩增的培养体系,为今后进一步研究北京油鸡骨骼肌卫星细胞提供技术平台。【方法】以15日龄的鸡胚胸肌为材料,采用联合酶消化法分离骨骼肌卫星细胞,差速贴壁法纯化细胞,使用Pax7、Desmin、Myod等骨骼肌卫星细胞的特异性标志对所得细胞进行免疫荧光鉴定,随后进行成肌诱导分化,并比较了3种培养体系对骨骼肌卫星细胞增殖的影响。【结果】细胞免疫荧光鉴定结果呈阳性,证实所培养的细胞为北京油鸡骨骼肌卫星细胞;成肌诱导后,细胞相互融合形成多核的肌管,成肌特异性标志MHC表达呈阳性;对不同扩增培养体系比较,结果表明,培养体系DMEM/F12+15%FBS+2.5ng·mL-1bFG最有利于北京油鸡骨骼肌卫星细胞的增殖。【结论】该试验成功地分离并鉴定了北京油鸡骨骼肌卫星细胞,建立了适于北京油鸡骨骼肌卫星细胞体外扩增的培养体系,同时成功地进行了成肌诱导分化,为今后研究北京油鸡骨骼肌生长和发育的机理提供了技术平台。

Isolation, Culture, Identification and Muscle Differentiation of Skeletal Muscle Satellite Cells in Beijing Fatty Chicken

【Objective】 This research was aimed to explore a protocol of isolation, culture and identification of skeletal satellite cells, and to establish an optimal in vitro amplification system for Beijing Fatty chicken skeletal muscle satellite cells, thereby laying a technical foundation for future pertinent researches. 【Method】 Pectoral muscles were isolated from 15-day-old chicken embryos, disassociated with collagenase and trypsin and purified via differential adhesion. Satellite cells were detected for the expression of Pax7, Myod and Desmin and induced to muscle. Proliferative capacities in three different culture systems were compared as well. 【Result】 The cultured cells expressed Pax7 and MyoD in nucleus and desmin in cytoplasm, demonstrating that they were indeed Beijing Fatty chicken skeletal muscle satellite cells. Satellite cells underwent muscle differentiation and formed multinucleated myotubes. Muscle differentiation markers MHC in cytoplasm were detected in these cells. And it represented an optimal system for Beijing Fatty chicken skeletal muscle satellite cells to be cultured in media containing DMEM/F12+ 15%FBS+2.5ng?mL-1 basic fibroblast growth factor (bFGF). 【Conclusion】 The present study successfully isolated and identified the skeletal muscle satellite cells in Beijing Fatty chicken and established their optimal in vitro culture system. Satellite cells were also successfully induced to differentiate into muscle , and, accordingly, provided a theoretical and technical basis for studying the mechanism of skeletal muscle growth and development in future.