Bcl-2 siRNA联合奥沙利铂对HT-29结肠癌细胞系凋亡的影响

Influence of RNA interference targeting at bcl-2 combined with oxaliplatin treatment on apoptosis of HT-29 cells

目的探讨bcl-2 siRNA联合奥沙利铂对结肠癌细胞HT-29凋亡的影响及相关作用机制。方法将化学合成的针对bcl-2的siRNA序列转染至HT-29细胞,将HT-29细胞分为5组:空白对照组、阴性对照组、干扰组、奥沙利铂组、联合组(干扰+奥沙利铂)。采用MTT比色法、Western blot、流式细胞术和分光光度法分别检测HT-29细胞增殖、Bcl-2和Bax蛋白表达、细胞凋亡及半胱氨酸天冬氨酸蛋白酶家族成员Caspase 9和Caspase 3的活化程度。结果 Bcl-2siRNA转染后,相对于奥沙利铂处理组,联合组细胞死亡率明显升高(P<0.01);Bax蛋白表达、Caspase 9和Caspase 3的活化程度显著升高(P<0.01)。结论 Bcl-2靶向RNA干扰联合奥沙利铂处理通过调节促凋亡蛋白表达从而促进了细胞凋亡,bcl-2可作为结肠癌基因治疗的后选新靶点。

Objective To explore the influence of RNA interference（ RNAi） targeting at bcl-2 combined with oxaliplatin treatment on apoptosis of HT-29 cells and its related mechanism. Methods The siRNA sequence targeting at bcl-2 which was synthesized by chemical method was transfected into HT-29 cells. The HT-29 cells were divided into five groups： blank control group,negative control group,siRNA group,oxaliplatin treated group and combination group（ siRNA combined with oxaliplatin treatment）. Then MTT assay,western blot,flow cytometry and spectrophotometry were employed to detect variations of cell viability,the expression of Bcl-2 and Bax,apoptosis,Caspase 9and Caspase 3 activity. Results After transfection,with compare to the oxaliplatin treated group,the rate of cell death in combination group were markedly increased（ P 〈 0. 01）; The expression of Bax,activities of Caspase 9 and Caspase 3 were obviously induced（ P 〈 0. 01）.Conclusion Bcl-2 siRNA combined with oxaliplatin treatment can induce cell apoptosis via upregulating apoptotic protein expression,bcl-2gene may be a candidate target in the gene therapy of colon cancer.