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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 investigate the role of resveratrol in inhibiting the growth of human glioma cell line U87 and inducing its apoptosis and activating caspase-3. Methods MTT method was used to draw the cell growth curve at different concentrations of Res for 6 h, 24 h and 48 h. Flow cytometry Annexin V-FITC and PI double staining were used to detect the apoptosis rate. Hoechst 33342 Fluorescence staining and transmission electron microscopy (TEM) were used to observe the changes of cell proliferation. The changes of caspase-3 were detected by Western-blot, the changes of caspase-3 mRNA were detected by semi-quantitative RT-PCR and the relative activity of caspase-3 by colorimetry. Results Res significantly inhibited the proliferation of U87 cells (P <0.01) in a concentration-dependent and time-dependent manner. Res also induced apoptosis in a concentration-dependent manner in U87 glioma cells. U87 cells were treated with different concentrations of Res for 24 hours. With the increase of drug concentration, the protein level of caspase-3 reduced and the level of caspase-3 mRNA increased (P <0.01). The cells were treated with 200 μmol / L Res for 0.5, 2, 6, 12 and 24 h respectively. The activity of caspase-3 began to rise at 2 h and reached the peak at 12 h (P <0.01) At 12 h after treatment, caspase-3 activity increased in a concentration-dependent manner (P <0.01). Conclusion Res significantly inhibits the growth of U87 cells and induces its apoptosis. Activation of caspase-3 is involved in the process of apoptosis.