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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

中国农业科学 2010,43(22):4725-4731 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2010.22.021

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

李方华

1,2

,侯玲玲,马月辉,庞全海,关伟军

3212

(1山西农业大学动物科技学院,山西太谷 030801;2中国农业科学院北京畜牧兽医研究所,北京 100193;3北京交通大学生命科学与生物工程

研究院,北京 100044)

摘要:【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。

关键词:北京油鸡;骨骼肌卫星细胞;鉴定;诱导分化

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

LI Fang-hua1,2, HOU Ling-ling3, MA Yue-hui2, PANG Quan-hai1, GUAN Wei-jun2

(1College of Animal Science and Technology, Shanxi Agriculture University, Taigu 030801, Shanxi; 2Institute of Beijing Animal Science and veterinary, Chinese Academy of Agricultural Sciences, Beijing 100193; 3Institute of Biological Science and Technology,

College of Science, Beijing Jiaotong Univeisity, Beijing 100044)

Abstract: 【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 收稿日期:2010-03-14;接受日期:2010-06-28

基金项目:国家科技支撑项目(2006BAD13B08、2008BADB2B01)、国家“863”计划项目(2006AA10Z198、2007AA10Z170)、转基因生物新品种

培育科技重大专项(2008ZX08009-003)

作者简介:李方华,硕士。Tel:137168-5410;E-mail:254711802@http://doc.xuehai.net。通信作者庞全海,教授,博士。E-mail:pangquanhai@http://doc.xuehai.net。并列

通信作者关伟军,教授,博士。Tel:010-62815992;E-mail:wjguan86@http://doc.xuehai.net

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