神经胶质瘤U251细胞氧化损伤中自噬和凋亡过程的时间顺序

The time order of autophagy and apoptosis process in the oxidative injury in glioma U251 cells

目的:探讨过氧化氢(H2O2)诱导神经胶质瘤U251细胞损伤中自噬和凋亡发生的时间顺序。方法:实验分为4组:正常对照组、1 mmol/L H2O2作用(6 h、12 h、24 h)组。应用MTT法检测H2O2对神经胶质瘤U251细胞生存率的影响;MDC染色检测自噬空泡的变化;流式细胞仪检测细胞凋亡率变化。Western blot检测Beclin 1和胞浆cyt c蛋白的表达。结果:与对照组相比,1 mmol/L H2O2作用下,U251细胞存活率明显降低,并呈时间依赖性。与对照组相比,1 mmol/L H2O2作用后,6 h时U251细胞自噬空泡明显增加,自噬相关蛋白Beclin 1表达明显增加,12 h、24 h细胞自噬水平逐渐增强;而6h时未见细胞凋亡率明显变化及cyt c由线粒体向胞浆的释放,12 h、24 h时细胞凋亡率明显增加,胞浆中cyt c蛋白表达明显增强(P<0.05)。结论:氧化损伤能够诱导神经胶质瘤U251细胞发生自噬和凋亡,并且自噬发生于凋亡之前。

Objective： To investigate the time order of autophagy and apeptosis in human U251 cells injury after H2O2 treatment. Methods： 4 groups in this study were set up, normal control group, 1 mmol/L H2O2（6 h,12 h,24 h） group. The viability of U251 cells treated with H2O2 was measured by MTr assay. Cell apoptotic ratio was determined by flow cytometry analysis. Autophagic vacuoles were stained with monodansylcadaverine. The protein level of Beclin 1 and cytosolic cyt c were assayed by using Western blot. Results： Compared with the control group, cell viability decreased significantly under 1 mmol/L H2O2 treatment in time-dependent way. Autophagic vacuoles and the expression of autophagic protein Beclin 1 increased at 6h, but cell apoptotic ratio and cytosolic cyt c protein did not change obviously, cell apoptotic ratio and cytosolic cyt c protein level increased at 12h and 24h（ P 〈 0.05）. Conclusion： Oxidative stress induced autophagy and apeptosis in U251 glioma cells, and autophagy eventuated ahead of apoptosis.