利用单根肌纤维法分离和培养猪骨骼肌卫星细胞及其成肌诱导分化_刘月光.pdfVIP

农业生物技术学报，2011 年，第19 卷，第5 期，第856~863 页 Journal of Agricultural Biotechnology, 2011, Vol.19, No.5, 856~863 研究报告 A Letter 利用单根肌纤维法分离和培养猪骨骼肌卫星细胞及其成肌诱导分化 刘月光 史新娥 沈清武 袁媛 杨秋梅 高晓娟 陈宗正 杨公社* 西北农林科技大学动物科技学院动物脂肪沉积与肌肉发育实验室，杨凌712100 * 通讯作者，gsyang999@ 摘 要 建立单根肌纤维法体外培养猪骨骼肌卫星细胞的体系，了解其增殖和成肌特性。通过 Ⅰ型胶原酶 消化，从猪骨骼肌中分离完整的单根肌纤维并培养，用细胞免疫荧光鉴定肌纤维上的卫星细胞，随后对从单 根肌纤维上游离出来的卫星细胞进行细胞免疫荧光染色，传代培养，成肌诱导分化和Western blot 分析骨骼 肌卫星细胞成肌特异性蛋白的表达。结果显示：分离并培养的单根肌纤维上附着有卵圆形的细胞，并随时间 的推移，细胞缓慢向外迁移并增殖，卫星细胞特异性标志基因对盒转录因子(Paired protein box, Pax7)和成肌 分化抗原(Myogenic Differentiation Antigen, MyoD)免疫荧光染色呈阳性，且阳性率达到90% 以上。成肌诱导 分化后，细胞开始汇合，并呈方向性生长，最终形成多核肌管,且成肌特异性标志基因Myogenin 和myosin heavy chain(MyHC)表达呈阳性。MyoD 蛋白高表达于增殖期，而Myogenin 和MyHC 在进入分化期才表达。 该实验成功建立了猪骨骼肌单根肌纤维的体外培养方法并获得了高纯度的卫星细胞，为骨骼肌卫星细胞进 行活体移植治疗相关疾病研究提供了实验材料。 关键词 骨骼肌纤维，卫星细胞，Pax7 ，猪 Isolation and Culture of Porcine Skeletal Muscle Satellite Cells by Single Muscle Fiber and Their Muscle Differentiation Liu Yueguang Shi Xine Shen Qingwu Yuan Yuan Yang Qiumei Gao Xiaojuan Chen Zongzheng Yang Gongshe* Laboratory of Animal Fat Deposition and Muscle Development, Northwest Agricultural and Forestry University, Yangling 712100, China * Corresponding author, gsyang999@ DOI: 10.3969/j.issn.1674-7968.2011.05.011 Abstract The present study established a porcine satellite cells in vitro model by means of the single fiber method to clarify the proliferation and myogenesis. Satellite cells can dissociate from the myofibers which isolated from porcine extensor digitorum longus (EDL) after digesting by collagenase Ⅰ. Then the cells were detected by their immunocytochemistry, growth curve, myogenesis induction and Western blot. The results showed that the cells adhered to the myofibers originally and then moved outside. More than 90 percent of the cells expressd the marker genes paired protein box(Pax7) and myogenic differentiation antigen(MyoD). After myogenesis induction, satellite cells began to convergence ,show