Objective To establish the methods of isolation,purification and culture of primary mouse myoblast,then to induce its myogenic differentiation and to detect the expression of microRNA-1(miRNA-1) and miRNA-155 during this process. Methods The primary mouse myoblast was isolated and purified from the hind leg of neonatal mouse with collagenase digestion , and difference of cell adherence. Its myogenic differentiation was induced by the differentiation culture medium containing horse serum. The myogenic ability of primary myoblast was identified by myosin heavy chain antibody (MF20) immunofluorescence staining. The expression of miRNA-1 and miRNA-155 during the myogenic differentiation was detected by Taqman real time PCR. Result The primary mouse myoblast was successfully isolated and purified;the myogenic differentiation of primary myoblast was successfully induced and MF20 immunofluorescence staining was positive. The miRNA-1 expression was dramatically up-regulated, while the miRNA-155 expression was down-regulated during the myogenic differentiation of primary mouse myoblast ,the difference was statistically significant(P<0.05). Conculsion The isolation and purification method of primary mouse myoblast is successfully constructed by this study ,which provides a model in vitro for investigating the miRNA gene regulation during myo-genic differentiation process.%目的建立小鼠原代成肌细胞分离、纯化及培养方法，并诱导其成肌分化，检测成肌分化过程中微小RNA-1（miRNA-1）和miRNA-155表达情况。方法提取新生小鼠后腿肌肉，经胶原酶消化，利用细胞贴壁差异性分离及纯化原代成肌细胞。使用含马血清的分化培养基诱导其成肌分化，通过肌球蛋白重链抗体———MF20免疫荧光染色鉴定成肌能力。使用Taqman实时定量聚合酶链反应检测原代成肌细胞分化过程中miRNA-1和miRNA-155表达情况。结果成功分离并纯化小鼠原代成肌细胞；成功诱导原代成肌细胞向骨骼肌分化，MF20免疫荧光染色呈阳性。原代成肌细胞在成肌分化过程中miRNA-1表达明显上升，miRNA-155表达明显下降，差异均有统计学意义（P<0.05）。结论成功构建了提取小鼠原代成肌细胞的方法，为进一步研究成肌分化过程中miRNA基因调控提供了体外模型。
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