Establishment of Rat Myoblast Atrophy Model Induced by Ang Ⅱ
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摘要:
目的 构建Ang Ⅱ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Ang Ⅱ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Ang Ⅱ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。0.1～10 μM的Ang Ⅱ对L6细胞活性无影响，而Western Blot和免疫荧光检测提示在5 μM和10 μM的Ang Ⅱ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10 μM Ang Ⅱ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。 Abstract:Objective To provide a model basis for further study of heart failure complicated with sarcopenia, a rat myoblast （L6） atrophy model induced by Ang Ⅱ was 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 Ang Ⅱ on L6 cells. To determine the conditions of the cell atrophy model, different concentrations of Ang Ⅱ were 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. Ang Ⅱ at 0.1 μm to 10 μm 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 10 μM Ang Ⅱ for 72 h. Conclusions The model of L6 cell atrophy can be established by the treatment with 10 μM Ang Ⅱ for 72 h. This model will provide the basis for in vitro cell study of heart failure complicated with sarcopenia. -
Key words:
- L6 cell /
- Heart failure /
- Sarcopenia /
- Skeletal muscle atrophy model
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图 1 不同时间下马血清诱导分化后L6细胞的形态（×100，100 μm）
Figure 1. Morphology of L6 cells after differentiation induced by horse serum at different times （×100，100 μm）
图 2 不同时间下马血清诱导分化后L6细胞MyHC蛋白的表达
Figure 2. Expression of MyHC protein in L6 cells after differentiation induced by horse serum at different times
图 3 不同浓度不同时间Ang Ⅱ对细胞活力的影响
Figure 3. Effects of Ang Ⅱ on cell viability at different concentrations and time
图 4 不同浓度Ang Ⅱ诱导下的细胞形态（×100，100 μm）
Figure 4. Cell morphology induced by different concentrations of Ang Ⅱ （×100，100 μm）
图 5 不同浓度Ang Ⅱ诱导下MyHC蛋白的表达
与分化组比较，**P < 0.01。
Figure 5. MyHC protein expression induced by different concentrations of Ang Ⅱ
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