疣毒净诱导H8细胞凋亡作用机制

目的:观察疣毒净制剂对人宫颈永生化细胞H8细胞增殖和凋亡的影响,探讨其可能的作用机制。方法:以转染HPV16基因后稳定传代的人宫颈永生化细胞株H8细胞为研究对象;经1,2,3,4,5 g·L~(-1)疣毒净干预H8细胞24,48,72 h,同时设立空白组,四甲基偶氮唑盐(MTT)比色法检测细胞增殖抑制作用;荧光显微镜下观察1,2,3,4,5 g·L~(-1)疣毒净干预24 h后,经Hoechst 33342染色的细胞凋亡情况;流式细胞仪检测1,2,3,4,5 g·L~(-1)疣毒净干预24 h后,经Annexin V/碘化丙啶(PI)双染后细胞早期凋亡率;蛋白免疫印迹法(Western blot)检测1,2,3,4,5 g·L~(-1)疣毒净干预24 h后H8细胞半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),聚腺苷二磷酸-核糖聚合酶(PARP)蛋白表达情况。结果:与空白组比较,2,3,4,5 g·L~(-1)疣毒净对H8细胞的生长抑制呈明显的浓度-时间依赖效应(P0.05);经Hoechst 33342染色后,荧光显微镜下可见1,2,3,4,5 g·L~(-1)疣毒净给药后细胞均出现明显的核染色质浓缩,3,4,5 g·L~(-1)疣毒净干预下圆形亮蓝色凋亡小体逐渐增多;流式细胞仪检测到1,2,3 g·L~(-1)疣毒净干预24 h后细胞的早期凋亡率分别为(23.73±5.72)%,(63.9±3.59)%,(71.53±1.59)%,与空白组(6.53±0.85)%比较早期凋亡率显著升高(P0.01);与空白组比较,疣毒净组凋亡执行蛋白Caspase-3及其底物PARP蛋白均明显下调(P0.05),呈浓度下调趋势,二者下调的趋势一致。结论:疣毒净能明显抑制宫颈永生化细胞H8细胞增殖,诱导早期凋亡,可能是通过激活Caspase-3-PARP途径来执行的。

Mechanism of Youdujing in Inducing Apoptosis of H8 Cells

Objective: To observe the effects of Youdujing preparation on the proliferation and apoptosis of cervical immortalized cells (H8 cells) and explore the possible mechanism. Method: H8 cells, which were transfected with HPV-16 gene and passed stably, were chosen to be the object of the study. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was used to detect the proliferation inhibition of H8 cells after being treated with 1, 2, 3, 4, 5 g·L^-1 Youdujing for 24, 48, 72 hours. Meanwhile, the blank was set up for parallel control. MTT was used to detect the inhibitory effect on cell proliferation. Apoptotic changes of cells that were intervened by 1, 2, 3, 4, 5 g·L^-1 Youdujing for 24 h and stained by Hoechst 33342 were observed by fluorescence microscope. Flow cytometry was used to detect the early-stage apoptotic rate of H8 cells after being double stained with Annexin V/propidium iodide (PI). Expressions of Caspase-3 protein and Poly ADP-ribose polymerase (PARP) protein in H8 cells, which were treated with 1, 2, 3, 4, 5 g·L^-1 Youdujing for 24 h, were tested by Western blot method. Result: The growth of H8 cells intervened by 1, 2, 3, 4, 5 g·L^-1 Youdujing was significantly inhibited in concentration-dependent and time-dependent manners (P<0.05), compared with blank group. According to Hoechst 33342 staining, nuclear chromatin condensations were observed after being intervened by 1, 2, 3, 4, 5 g·L^-1 Youdujing for 24 h, and round, bright blue apoptotic bodies increased after being treated with 3, 4, 5 g·L^-1 Youdujing. The early-stage apoptotic rates of cells treated with 1, 2, 3 g·L^-1 Youdujing were (23.73±5.72)%,(63.9±3.59)%,(71.53±1.59)%, respectively, according to flow cytometry, which were significantly higher than that of control group(6.53±0.85)%(P<0.01). Caspase-3 and PARP proteins were consistently down-regulated in H8 cells (P<0.05). Conclusion: Youdujing preparation can inhibit H8 cell growth and induce early-stage apoptosis in H8 cells, which may be implemented by activating Caspase-3-PARP.