失神经萎缩肌组织成分对成肌细胞性能影响的实验研究

Experimental study of the features of myoblast treated with component of long-term denervated muscle tissue

目的:探讨长期失神经萎缩骨骼肌组织成分对体外培养成肌细胞的性能影响。方法:①获取长期失神经支配肌萎缩模型大鼠的腓肠肌,无菌条件下清洗、剪碎、匀浆、过滤,收集上清液;②收集Schwann细胞的体外培养基;③体外培养C2C12成肌细胞系,扩增传代并分组:A.对照组,常规培养,无任何处理因素;B.添加Schwann细胞培养基;C.添加萎缩肌匀浆液。倒置显微镜观察3组细胞的形态特征,免疫荧光检测C2C12细胞MyoD和Myogenin的差异表达。结果:倒置显微镜下,各组细胞贴壁良好,A、C组形态无差异,未见分化肌管;B组于培养48h后细胞变纤细,可见肌管形成。荧光染色证实,培养24h后,3组均出现MyoD阳性细胞,C组阳性细胞数量明显多于A、B组;72h时,3组均可见Myogenin阳性细胞,B组阳性细胞数量明显多于A组,C组阳性细胞数量稀少。结论:长期失神经萎缩肌组织成分能够促进成肌细胞的增殖与活化,但抑制活化成肌细胞的进一步分化成熟。

Objective： To explore the influence of component of denervated muscle tissue on the features of myoblast cultured in vitro. Methods： Gastrocnemius collected from model rat suffering from long-term denervation was homogenated under sterile condition. Supernatant fluid of denervated muscle was added into C2C12 myoblasts cultured in vitro. After 24 and 48 hours, the morphologic features, the expression of MyoD and Myogenin in myoblasts were detected by inverted microscope and immunofluorescence technique respectively. Meanwhile, normal cultured cells and those treated with medium of Schwann cell were taken as negative and positive control respectively.Results：Cultured cells grew well in vitro and there were no morphologic difference between negative control and denervation group. The C2C12 myoblast of positive control showed thin shape and formed tiny myotubes. After 24 hours of cultures, MyoD positive cells appeared in every group, however, the number of positive cells in denervation group was more than another two groups. Myogenin positive cells also were found in every group at 72 hours, with the most number of positive cells in positive group, and the least number in denervation group. Conclusions： component of long-term denervated skeletal muscle will promote the proliferation and activation of myoblast cultured in vitro, as well inhibit differentiation of activated cells.