Bax/Bak在抑制人颗粒细胞凋亡中的作用

Inhibitory effect of RNA interference targeting BaxBak on apoptosis of human granulosa cells

目的探讨Bax、Bak等凋亡分子的小分子干扰RNA(siRNA)可否抑制人颗粒细胞凋亡。方法(1)用Western blotting检测Bax-siRNA、Bak-siRNA;(2)将Bax-siRNA、Bak-siRNA分别或同时转染入人颗粒细胞,观察基本形态,用流式细胞仪检测颗粒细胞的凋亡。结果(1)转染了Bax-siRNA和Bak-siRNA后的条带减弱,说明细胞内Bax、Bak含量降低。(2)空白对照组、Bak干扰组、Bak+Bax干扰组细胞形态正常;Bax干扰组细胞形态基本正常,胞质中有少数黑点;阳性对照组、阴性对照组细胞变圆,收缩,细胞间距大,胞质中有较多黑点。(3)流式细胞仪检测结果:Bak干扰组凋亡指数为3.44%,Bax+Bak联合干扰组凋亡指数为3.97%,明显低于阴性对照;Bax干扰组凋亡指数为19.98%,与阴性对照组相似。结论(1)干扰Bak等凋亡分子的表达,可以抑制颗粒细胞的凋亡。(2)Bak干扰组及Bax+Bak联合干扰组抗颗粒凋亡效果显著。(3)Bax干扰组抑制颗粒细胞凋亡的作用不明显。

Objective To investigate the inhibitory effect of small interfering RNA （siRNA） targeting Bax-Bak on the apoptosis of human granulosa cells. Methods Human granulosa cells were transfected with Bax-siRNA and Bak-siRNA either alone or in comibnation, and the cell morphological changes were obsered and the cell apoptosis was detected with flow cytometry. Western blotting was performed to examine the changes in Bax and Bak expressions in the transfected cells. Results Western blotting demonstrated significantly weakened expressions of Bax and Bak in the transfected cells. The cell morphology of the cells tranfected with Bak siRNA and with both Bak and Bax siRNA remained normal; the cells with exclusive Bax siRNA transfection presented with basically normal cell morphology, but black spots were noted in the cytoplasm. In the positive and negative control groups, the cells became rounded and shrank with expanded intercellular spaces and numerous black spots in the cytoplasm. Flow cytometry showed apoptotic indexes of 3.44% and 3.97% in cells transfected with Bak siRNA and Bax-Bak siRNA, respectively, significantly lower than that in the negative group. Bax siRNA transfection resulted in an apoptotic index of 19.98%, similar to that in the negative group. Conclusion Interference of the expression of Bak gene inhibits the apoptosis of human granulosa cells, and the inhibitory effect can be enhanced by simultaneous Bax interference, which, when used alone, does not obviosuly inhibit the apoptosis of human granulosa cells.