白藜芦醇诱导U87胶质瘤细胞凋亡及caspase-3的激活

U87 glioma cell apoptosis and caspase-3 activation induced by resveratrol

目的探讨白藜芦醇(Res)抑制体外脑胶质瘤细胞系U87生长并诱导其凋亡,激活 caspase-3的作用。方法四甲基偶氮唑蓝(MTT)法绘制不同浓度Res作用6 h、24 h、48 h后的细胞生长曲线,流式细胞仪Annexin V—FITC和PI双染检测凋亡率,Hoechst 33342荧光染色及透射电镜 (TEM)观察细胞增殖改变:Western-blot分析caspase-3酶原的变化,半定量RT—PCR检测caspase-3 mRNA水平的改变,比色法测定caspase-3的相对活性。结果 Res明显抑制U87细胞的增殖 (P<0．01),呈浓度及时问依赖性反应;Res可诱导U87胶质瘤细胞凋亡并呈浓度依赖关系。用不同浓度 Res处理U87细胞24 h,随药物浓度增加,caspase-3酶原的蛋白水平减少,caspase-3 mRNA水平增加 (P<0．01)。用200 μmol／L Res分别处理细胞0．5、2、6、12、24 h,caspase-3活性于2 h开始升高,12 h达高峰(P<0．01);用不同浓度的Res处理细胞12 h,caspase-3活性呈浓度依赖性的升高(P<0．01)。结论 Res明显抑制U87细胞生长并诱导其发生凋亡,凋亡过程中有caspase-3的激活。

Objective To explore the effect of resveratrol （Res） on the proliferation and apoptosis of brain glioma cell line U87 in vitro and the activation of caspase-3. Methods Methyl thiazolyl tetrazolium （MTT） assay was used to test the changes in the proliferation of U87 cells cultured with different concentrations of Res for 6, 24 and 48 h and accordingly draw a cell growth curve. The Annexin V-FITC and PI stainings were applied to detect the apoptosis rate induced by Res by flow cytometer （FCM）. Hoechst 33342 fluorescence staining, transmission electron microscope （TEM） were applied to observe the changes of cell morphology. The changes in pro-caspase-3, caspase-3 mRNA and caspase-3 relative activity were analyzed by Western-blot, semi-quantitative RT-PCR and colorimetric assay, respectively. Results Res obviously suppressed the proliferation of U87 glioma cells （P〈0.01） in a concentration- and time-dependent manner. Res induced the apoptosis of glioma cells in a concentration-dependent manner. After U87 cells were treated with 0,25,50,200,400μmol/L Res for 24 h respectively,the level of pro-caspase-3 protein decreased and caspase-3 mRNA increased both as concentration increased （P〈0.01）. When the cells were treated with 200μmol/L Res for 0.5, 2, 6, 12, 24 h, the caspase-3 activity began to rise at 2 h, reached the peak at 12 h （P〈0.01）. After the cells were treated with Res of different concentrations for 12 h, caspase-3 activity increased in a concentration-dependent manner （P〈0.01）. Conclusion Resveratrol significant inhibits glioma cell proliferation and induces its apoptosis. Caspase-3 is activated during the Res-induced apoptosis of U87 cells.