不同代次牛成肌细胞的诱导分化及相关基因的表达分析

Myoblast Differentiation with Different Passages and Its MyoGenisis Related Gene Expression

利用初生荷斯坦牛的成肌细胞,在不同代次以含体积分数为2%马血清的DMEM进行诱导分化,在之后0、2、4、6、8、10d观察细胞的形态变化并收集细胞提取总RNA,检测成肌相关基因的表达情况,为进一步研究牛肌肉发生过程及相关基因的表达调控提供依据。同时利用实时荧光定量PCR分别检测肌性相关基因MyoD(生肌决定因子)、MyoG(肌细胞生成素)以及非肌性相关基因A-FABP(脂肪细胞型脂肪酸结合蛋白)表达水平的变化。研究表明:1各代细胞在诱导培养4d后开始有肌管形成,其后越来越多,8d时达高峰。2成肌细胞向成熟肌细胞分化过程中,MyoD、MyoG、A-FABP基因都呈先上升然后下降的趋势。3高代次成肌细胞比低代次细胞增殖慢,而且在诱导分化后,肌管的数目明显变少;MyoD、MyoG、A-FABP随着代次的升高而下降。故推断MyoD、MyoG以及A-FABP基因会在成肌分化开始后的不同阶段被激活,从而发挥不同的调控作用,而且随着传代次数的增加成肌细胞的分化能力减弱。

In this study, myoblasts were derived from new born calf bulls. The cells of every passage were cultured with DMEM containing volume fraction of 2 G horse serum and induced differentiation at the 0th, 2nd, 4th, 6th, 8th, and 10th day, respectively. Morphological changes of the cells were observed under the phase contrast microscope, followed by cell collection. In addition, expressions of the muscle-related genes MyoD and MyoG as well as the non-muscle-related gene A-FABP were de- tected by real-time quantitative PCR. This research indicated that： （1） at the 4th day, cells began to form a muscular tube and the myotubes grew fast until the 8th day. （2） During the myoblast differenti- ation process, expressions of MyoD, MyoG, and A-FABP genes were in different levels, with stage- specific property. （3） The myoblasts of older passage had fewer cells than the younger passage in pro- liferation, and the number of muscle cells in differentiation also reduced significantly. Therefore, this research suggested that MyoD, MyoG, and A-F＇ABP were activated at the beginning of myoblast dif- ferentiation; the differentiation capacity of myoblast diminished with the increase of passage number.