周期性机械牵张刺激对C_2C_(12)小鼠成肌细胞增殖和有氧代谢能力的影响

Effects of cyclic mechanical stretch stimulation on proliferation and aerobic capacity of C_2C_(12) myoblasts

目的探讨不同频率牵张刺激对C_2C_(12)小鼠成肌细胞增殖及细胞有氧代谢能力的影响。方法采用Flexercell细胞应力加载装置对体外培养的C_2C_(12)成肌细胞分别加载1、2 Hz不同频率的牵张刺激,牵张幅度为15%,牵张时长为2 h/d,连续进行4 d。以静置培养的C_2C_(12)成肌细胞作为对照组。实验期间通过倒置相差显微镜观察细胞生长状态,利用CCK-8试剂盒测定细胞增殖情况。实验结束后,分别收取各组细胞,经胰酶消化处理,然后利用荧光探针,通过能量代谢设备检测细胞耗氧量比率。结果细胞经机械牵张刺激后,显微镜下细胞形态为典型的长梭形,排列呈现一定方向性,与牵张刺激方向平行,生长状况良好;与对照组细胞相比,1、2 Hz频率牵张刺激均可显著促进细胞增殖(P<0.05),且在第3、4 d,1 Hz实验组的增殖情况优于2 Hz实验组;实验组与对照组细胞耗氧量比率均有增加,但各组之间并无显著差异(P>0.05)。结论周期性机械牵张刺激能有效促进C_2C_(12)小鼠成肌细胞增殖,且与牵张刺激的频率有关,其中1 Hz的牵张频率最佳,但牵张刺激对C_2C_(12)小鼠成肌细胞的有氧代谢能力无明显影响。

Objective To investigate the influence of mechanical stretch at different frequencies on proliferation and aerobic capacity of mice myoblast cells C2C12. Methods C2C12 cells cultured in vivo were exposed to mechanical strain with the magnitude of 15% at the frequency of 1 Hz and 2 Hz,respectively,for 2 hours per day over a period of 4 days by using Flexercell Cell Tension System,while in control group C2C12 cells were cultured statically. The C2C12 cells were observed by inverted phase contrast microscope. CCK-8 Cell Counting Kit was used to estimate the proliferation of cells. After the experiment,the cells from both groups were obtained by trypsin digestion. Mito Xpress-Xtra system with oxygen sensitive probe was introduced to detect the extracellular oxygen consumption level. Results The morphology of C2C12 cells presented a typical long spindle under the microscope following the mechanical stretch stimulation. The cells were arranged in a certain direction,parallel to the direction of tension stimulation and growing in good condition. Compared with the control group,the cell numbers in both 1 Hz group and 2 Hz group were significantly increased（ P〈0. 05）. At the 3rd and 4th day,the number of cells in 1 Hz group significantly increased more than that in 2 Hz group. However,the oxygen consumption capacity increased in both experimental groups and the control group,but no significant difference was found in between（ P〉0. 05）. Conclusions Cyclic mechanical stretch stimulation can effectively induce proliferation of C2C12 cells,which is related to the frequency of mechanical stretch,with the frequency of 1 Hz being optimum. But stretch stimulation has no significant impact on the aerobic ability of C2C12 cells.