AngⅡ诱导大鼠成肌细胞萎缩模型的构建

Establishment of Rat Myoblast Atrophy Model Induced by AngⅡ

目的构建AngⅡ诱导大鼠成肌细胞(L6)萎缩模型,为进一步研究心衰合并肌少症提供模型依据。方法采用大鼠成肌细胞(L6),用2%马血清诱导分化,镜下观察和MyHC蛋白表达情况判断诱导分化的时间。CCK-8检测不同浓度AngⅡ对L6细胞的影响。为确定细胞萎缩模型的条件,选择不同浓度的AngⅡ作用于L6细胞,并用Western Blot和免疫荧光检测MyHC蛋白的表达情况。结果L6细胞在2%马血清下生长6 d,能被成功诱导分化,镜下显示细胞出现融合现象和肌管,MyHC蛋白表达也增加。0.1~10μM的AngⅡ对L6细胞活性无影响,而Western Blot和免疫荧光检测提示在5μM和10μM的AngⅡ作用72 h下,MyHC蛋白表达水平逐渐下降。结论诱导分化后的L6细胞经10μM AngⅡ刺激72 h可形成细胞萎缩模型,为心衰合并肌少症的体外细胞研究提供基础。

Objective To provide a model basis for further study of heart failure complicated with sarcopenia,a rat myoblast(L6)atrophy model induced by AngⅡwas established.Methods 2%horse serum was used to induce differentiation of L6 cells.The time of induced differentiation was determined by microscopic observation and MyHC protein expression.CCK-8 was used to detect the effects of different concentrations of AngⅡon L6 cells.To determine the conditions of the cell atrophy model,different concentrations of AngⅡwere selected to treat L6 cells,and the expression of MyHC protein was detected by Western Blot and immunofluorescence.Results L6 cells were induced to differentiate successfully after growing in 2%horse serum for 6 days.Microscopic observation revealed the fusion and myotubule of L6 cells,and the expression of MyHC protein was also increased.AngⅡat 0.1μm to 10μm had no effect on the activity of L6 cells.Western Blot and immunofluorescence detection showed that the MyHC protein expression level decreased after treated with 5 and 10μM AngⅡfor 72 h.Conclusions The model of L6 cell atrophy can be established by the treatment with 10μM AngⅡfor 72 h.This model will provide the basis for in vitro cell study of heart failure complicated with sarcopenia.