摘要
目的观察wortmannin和U0126抑制胰岛素对大鼠成肌细胞向成熟肌细胞分化作用,探索定向诱导肌分化的途径及其分子机制。方法分离和培养大鼠骨骼肌成肌细胞,用含不同浓度胰岛素的DMEM培养基培养,收集细胞,在相差显微镜下观察形态变化,并通过免疫细胞化学方法和Western blot法检测生肌素(myogenin)表达。用磷脂酰肌醇3激酶(PI3K)特异性抑制剂wortmannin和MEK特异性抑制剂U0126干预后,观察胰岛素对骨骼肌成肌细胞分化的影响。结果胰岛素能明显促进骨骼肌成肌细胞形成肌小管,诱导2 d开始有肌管形成并逐渐增多到7 d达到高峰;胰岛素促进骨骼肌成肌细胞的生肌素阳性细胞核和生肌素蛋白表达增多,而wortmannin和U0126能下调胰岛素的这种作用。结论 Wortmannin和U0126可阻断胰岛素促进骨骼肌成肌细胞向成熟肌细胞分化,使肌管生成减少,生肌素表达下调。
Objective To verify whether insulin promotes the differentiation of skeletal myoblasts into myocytes and whether wortmannin and U0126 inhibit the promoting effect with an attempt to explore the molecular mechanisms of inducing the differentiation of muscular cells in rats. Methods Primary skeletal myoblasts were separated and cultured from rats, and then were treated in DMEM containing various concentrations of insulin. The morphology of cells was monitored under a phase-contrast microscope. And the expression of myogenin was detected by immunocytochemistry and Westem blotting. The change in the effect of insulin on the differentiation of myoblasts was observed after the intervention of a phosphatidylinositide 3-kinase （PI3K） inhibitor wortmannin and a specific MEK inhibitor U0126. Results Insulin markedly promoted myotube formation of myoblasts. Two days after insulin treatment, myotubes started to form; later, more and more myotubes appeared, and to the peak at 7 days. Insulin increased the expression of myogenin in a concentration-dependent manner. However, wortmannin and U0126 inhibited the effect of insulin on the differentiation of skeletal myoblasts in rats. Conclusion Wortmannin and U0126 can suppress the promoting effect of insulin on the differentiation of skeletal myoblasts into myocytes in rats and decrease the formation of myotubes and the expression of myogenin.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第7期717-720,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
江西省教育厅重点科研项目(GJJ11694)
国家自然科学基金(81000075)
