Abstract：Myoblast is capable of self-renewal and polymorphism, and plays an important role in the regeneration and repair of skeletal muscle. In order to establish a method for isolation and purification, culture and identification of dairy goat (Capra hircus) myoblasts in vitro, and to understand the identification of myoblast features of proliferation and myogenesis, and to provide experimental materials for the regulation mechanism of muscle development in dairy goats, the samples were taken from skeletal muscle of Xinong Saanen dairy goat kids. Cells were disassociated with collagenase I and trypsin following with purification of cell using differential adhesion approach. qRT-PCR and immunofluorescence staining were conducted to further identify purified cells. Cell morphological results showed that purified cells were in spindle shape and maintained a stable status of growth. Myogenic regulatory factors, myogenic differentiation factor (MyoD) and myogenic regulatory factor 5 (Myf5), and desmin were positively expressed in purified cells (percentage of purified cell ＞ 95%) as observed from the results of qRT-PCR and immunofluorescence staining. Purified myoblasts were able to fuse into myotubes after induced differentiation. Myosin heavy chain (MYHC), a specific marker of differentiated myotubes, was positively expressed. This study establish a method of isolation and culture of dairy goat myoblasts in vitro. Myoblast line with high purity is successfully obtained, which is able to be used as an experimental material in studies of regulation mechanism of muscle development in dairy goats.
[1]刘立斌, 周红雨.成肌细胞的临床应用现状及前景[J].中国修复重建外科杂志, 2007, 21(7):767-770[2]李俊玲, 王世立, 韩金祥.肌源干细胞研究进展[J].中国生物工程杂志, 2007, 27(5):125-130[3]熊雷, 何衍佶, 戴威, 赵圆, 高彦飞, 邓忠良, 聂茂.小鼠原代成肌细胞分离、纯化及培养[J].现代医药卫生, 2017, 33(03):337-339[4]邱裕佳, 王伟.成肌细胞原代培养及临床应用前景[J].生物骨科材料与临床研究, 2017, 14(2):72-75[5]陈晓萍, 刘红, 吴燕, 等.成人成肌细胞快速高效分离方法的建立[J].中华神经医学杂志, 2003, 2(6):404-406[6]Rando T A, Blau H M.Primary mouse myoblast purification,characterization,and transplantation for cell-mediated gene therapy[J].Journal of Cell Biology, 1994, 125(6):1275-1287[7]Gharaibeh B, Lu A, Tebbets J, et al.Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique[J].Nature Protocols, 2008, 3(9):1501-9[8]王红娜, 张英杰, 刘月琴.绵羊成肌细胞的纯化、培养、鉴定及其成肌诱导分化研究[J].河北农业大学学报, 2016, 39(01):94-98[9]李伟.山羊骨骼肌卫星细胞的分离及成肌调节因子的克隆、表达与功能初探[D]. 扬州大学, 2013.[10]张玉石, 李汉忠, 张锐强, 等.应用分离纯化组织工程用肌卫星细胞[J].中国医学科学院学报, 2006, 28(2):182-185[11]陈立.大鼠肌卫星细胞体外培养及移植延缓失神经骨骼肌萎缩的实验研究[D]. 复旦大学, 2012.[12]Hindi L, Mcmillan J D, Afroze D, et al.Isolation, Culturing, and Differentiation of Primary Myoblasts fromSkeletal Muscle of Adult Mice[J].Bio-protoco, 2017, 7(9):1-17[13]安静.IGF1在绵羊成肌细胞增殖与分化中的作用[D]. 新疆农业大学, 2013[14]徐可, 方祖军, 李映川, 等.大鼠骨骼肌卫星细胞体外培养生长和成肌特性[J].上海交通大学学报医学版, 2008, 28(7):775-778[15]Diermeier S, Iberl J, Vetter K, et al.Early signs of architectural and biomechanical failure in isolated myofibers and immortalized myoblasts from desmin-mutant knock-in mice[J].Sci Rep, 2017, 7(1):1-3[16]刘文斌, 吴丽姿, 李睿, 等.新生小鼠骨骼肌成肌细胞分离和分化方法研究[J].武汉轻工大学学报, 2015, 34(1):21-25[17]Darmon M, Serrero G, Rizzino A, et al.Isolation of myoblastic,fibro-adipogenic,and fibroblastic clonal cell lines from a common precursor and study of their requirements for growth and differentiation ☆[J].Experimental Cell Research, 1981, 132(2):313-327[18]孙文娟.猪骨骼肌来源成肌和成脂细胞分化差异的分子机制[D]. 中国农业大学, 2017[19]冯青辉.肉羊骨骼肌卫星细胞的培养及MRFs基因表达量的分析[D].内蒙古农业大学, 2017[20]Relaix F, Zammit P S.Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage[J].Development, 2012, 139(16):2845-2856[21]Tabakov V Y, Zinov’Eva O E, Voskresenskaya O N, et al.Isolation and Characterization of Human Myoblast Culture In Vitro,for Technologies of Cell and Gene Therapy of Skeletal Muscle Pathologies[J].Bulletin of Experimental Biology & Medicine, 2018, 164(12):1-7[22]Cabe?o L C, Budri P E, Baroni M, et al.Maternal protein restriction induce skeletal muscle changes without altering the MRFs MyoD and myogenin expression in offspring[J].Journal of Molecular Histology, 2012, 43(5):461-471[23]李方华, 侯玲玲, 马月辉, 等.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[J].中国农业科学, 2010, 43(22):4725-4731
