自噬在应力诱导成肌细胞凋亡中的作用初探

The Role of Autophagy in Cyclic Stretching Induced Myoblast Apoptosis

目的:探讨自噬在周期性张应力介导的成肌细胞凋亡中的作用,以明确应力诱导内质网应激引起自噬与凋亡之间的关系。方法:在成功构建L6大鼠体外培养--力学刺激模型的基础上,采用Western Blot法分析周期性张应力对自噬相关蛋白LC3蛋白表达的影响,并通过Annexin V-FITC/PI流式细胞术检测细胞凋亡情况。加力组分别给予1,6,12,24 h的力学刺激(拉伸变形率为15%,频率为10循环/min),3-MA组和Rapamycin组在加力2 h前分别加入自噬抑制剂3-甲基腺嘌呤和自噬激活剂雷帕霉素并且加力24 h,0 h组与实验组在同时种板但是不给予力刺激。采用SPSS17.0统计软件对以上数据进行统计分析。结果:成肌细胞中的LC3II/LC3I值随加力时间延长呈上升趋势,24 h达最高(P<0.05);抑制组的细胞凋亡率(18.75±1.06%)相对于0 h组(0.726±0.13%)和加力24 h组(14.84±1.14%)的明显升高(P<0.05);Rapamycin组相对于加力24 h组的细胞凋亡率明显下降(8.88±1.08%vs 14.84±1.14%),但是细胞凋亡率仍然高于0 h组的(8.88±1.08%vs 0.726±0.13%)。结论:在一定时间范围内,周期性张应力可诱导成肌细胞发生自噬,并且自噬活性与作用时间成正比;自噬可以降低应力介导的成肌细胞凋亡的活性。

Objective： To study the function of autophagy in which cyclic stress induced-apoptosis in myoblasts and clarity the relationship between autophagy and apoptosis under the mechanical stress.Methods： Based on the mechanical stimulation model of myoblasts were constructed successfully in vitro,to analysis effects of cyclic tensile stress of LC3 protein expression by Western Blot.Flow cytometric analysis of the stretched cells for Annexin V-FITC/PI staining was performed to detect apoptotic cells.Continuous cyclic stretching for 1,2,6,12 and 24 hours（15 % surface elongation at a frequency of 10 cycles/min）,the inhibitor group which 3-methyladenine（3-MA） and the Rapamycin group was added to myoblasts 2 h before cyclic stretch,and then 24 h stretch was applied.The 0 h group which without mechanical stimulation and the experimental group were cultured at the same time.Using SPSS17.0 statistical software for statistical analysis of the above data.Results： The ratio of LC3 II /LC3 I increased with the loading time prolonged and the 24 h reached the highest（compared with the control group,the differences were significant difference,P〈0.05）.The ratio of apoptotic cells in the3-MA group（18.75±1.06 %） is higher than loading 24 h group（14.84±1.14 %）; the ratio of apoptotic cells in Rapamycin group is lower than loading 24 h group but higher than 0 h group（8.88±1.08 % vs 14.84±1.14 %,8.88±1.08 % vs 0.726±0.13 %）.Conclusion：Cyclical stretch could induce autophagy in myoblasts,and autophagy activity was increased with the loading time prolonged and the 24 h reached the highest.Autophagy can decrease the activity of cyclical stretch-apoptosis.