澳洲茄边碱诱导胶质母细胞瘤U251细胞凋亡的机制研究

Mechanism of Solamargine-Induced Apoptosis in Glioblastoma U251 Cells

目的 探讨澳洲茄边碱(SM)对胶质母细胞瘤U251细胞凋亡的影响及作用机制。方法 用SM(0,2,4,6,8μmol/L)处理细胞24 h,即溶剂对照组和SM 1组、2组、3组、4组。采用CCK-8法和克隆形成实验分别检测细胞活力和增殖能力,并计算细胞存活率和克隆形成率;采用Annexin V-FITC/PI双染色法及流式细胞术检测细胞凋亡情况,并计算细胞凋亡率;采用蛋白免疫印迹(Western blot)法检测凋亡相关蛋白表达水平;采用2’,7’-二氯荧光素二乙酸酯(DCFH-DA)和JC-1荧光探针分别检测U251细胞中活性氧水平和线粒体膜电位。结果 与溶剂对照组比较,U251细胞存活率和克隆形成率随SM浓度的增加均呈降低趋势(P <0.05),细胞凋亡率呈升高趋势(P <0.01);经SM处理后,细胞剪切多聚腺苷二磷酸核糖聚合酶、剪切胱天蛋白酶3蛋白表达水平及细胞内活性氧水平均呈升高趋势(P <0.05),线粒体膜电位呈降低趋势(P <0.01)。结论 SM可能通过激活线粒体介导的细胞凋亡通路,从而抑制胶质母细胞瘤U251细胞活性。

Objective To investigate the effect and mechanism of solamargine(SM)in inducing apoptosis of glioblastoma U251 cells.Methods U251 cells were treated with SM(0,2,4,6,8μmol/L)for 24 h,which were taken as the solvent control group and SM 1,2,3,and 4 groups respectively.CCK-8 assay and clone formation experiment were used to detect cell viability and proliferation ability respectively,and the cell survival rate and clone formation rate were calculated.Annexin V-FITC/PI dual staining method and flow cytometry were used to detect cell apoptosis,and the apoptosis rate was calculated.The expression level of apoptosis-related proteins was detected by the Western blot.The reactive oxygen species levels and mitochondrial membrane potential levels in U251 cells were observed by 2',7'-Dichlorofluorescein diacetate(DCFH-DA)and JC-1 fluorescent probes respectively.Results Compared with those in the solvent control group,the survival rate and clone formation rate of U251 cells showed a decreased trend with the increase of SM concentration(P<0.05),while the apoptosis rate of U251 cells showed a increased trend with the increase of SM concentration(P<0.01).After SM treatment,the expression levels of cell cleaved-poly adenosine diphosphate ribose polymerase(C-PARP),and cleaved-caspase 3(C-caspase 3)protein,and intracellular reactive oxygen species levels showed a increased trend(P<0.05),and mitochondrial membrane potential showed a decreased trend(P<0.01).Conclusion SM may inhibit the activity of glioblastoma U251 cells by activating the mitochondrial-mediated apoptosis pathway.