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安淮山羊骨骼肌卫星细胞的分离培养与鉴定
郑琪,睢梦华,朱龙,吴昊,丁建平,张运海,凌英会
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(安徽农业大学动物科技学院,合肥 230036; 安徽地方畜禽遗传资源保护与生物育种省级实验室,合肥 230036;安徽农业大学动物科技学院,合肥 230036; 安徽地方畜禽遗传资源保护与生物育种省级实验室,合肥 230036; 安徽省羊繁育工程技术研究中心,合肥 230036)
摘要:
为了分离得到安淮山羊骨骼肌卫星细胞,成功构建山羊骨骼肌卫星细胞系,为相关研究提供实验材料。以安淮山羊背最长肌肌肉组织为材料,采取胶原酶和胰蛋白酶结合的二步消化法,结合差速贴壁法纯化原代骨骼肌卫星细胞。对骨骼肌卫星细胞中特异性基因Pax7用免疫荧光染色法及PCR扩增加以鉴定。通过分离、纯化,得到纯度较高的山羊骨骼肌卫星细胞,其形态为圆形,折光性强。培养一段时间后密度增大,细胞呈梭型。所得骨骼肌卫星细胞生长状态良好,具有较为一致的生长方式和形态特点。经免疫荧光染色法以及PCR鉴定发现细胞系中有Pax7基因的表达。通过上述材料和方法,可获得较纯的山羊骨骼肌卫星细胞,并稳定传代。
关键词:  山羊  骨骼肌卫星细胞  细胞分离  体外培养  免疫荧光染色
DOI:10.13610/j.cnki.1672-352x.20170419.026
基金项目:安徽省自然科学基金项目(1708085MC61)和安徽省现代农业产业技术体系(2016-2020)共同资助。
Separation and identification of Anhuai goat skeletal muscle satellite cells
ZHENG Qi,SUI Menghua,ZHU Long,WU Hao,DING Jianping,ZHANG Yunhai,LING Yinghui
(School of Animal Science and Technology, Anhui Agricultural University, Hefei 230036; Local Animal Genetic Resources Conservation and Biobreeding Laboratory of Anhui Province, Hefei 230036;School of Animal Science and Technology, Anhui Agricultural University, Hefei 230036; Local Animal Genetic Resources Conservation and Biobreeding Laboratory of Anhui Province, Hefei 230036; Engineering Research Center of Reproduction and Breeding in Sheep of Anhui Province, Hefei 230036)
Abstract:
The skeletal muscle satellite cells of Anhuai goat were isolated and used for construction of a series of experimental materials. The skeletal muscle satellite cells from longissimus muscle tissue of goats were isolated by collagenase and trypsinase digestion method. The satellite cells were purified by differential adhesion and cultured by passage, cryopreservation and other experimental methods. The marker gene of paired protein box (Pax7) of the cells was detected by PCR and immunocytochemistry technology. After purified by differential adhesion method, the high purity skeletal muscle satellite cells were grown from sphere with a strong refraction to long-shuttle in shape after culturing for a certain time. The results showed that the isolated muscle satellite cells were healthy and had a similar growth style and morphological characteristics. The expression of Pax7 in the cells was detected through immunofluorescent staining and PCR amplication. The analysis result showed that high purity skeletal muscle satellite cells were obtained and these cells were genetic stable.
Key words:  goat  skeletal muscle satellite cells  cell isolation  cell culture  immunocytochemistry technology

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