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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(P<0.05);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(P<0.01);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(P<0.05),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。
OBJECTIVE: To observe the effects of wududujing preparations on the proliferation and apoptosis of human immortalized human H8 cells and to explore its possible mechanism. Methods: Human cervical immortalized cell line H8 stably passaged after transfection of HPV16 gene was used as the research object. After being treated with 1, 2, 3, 4, 5 g · L -1 wart virus, H8 cells 24, 48 , 72 h respectively. At the same time, the blank group was established and MTT colorimetric assay was used to detect the cell proliferation inhibitory effect. The cells of 1, 2, 3, 4, 5 g · L -1 24 h after poisoning, the apoptosis of cells stained with Hoechst 33342 was detected by flow cytometry. After the cells were treated with 1, 2, 3, 4, 5 g · L -1 wart virus for 24 h, / Propidium iodide (PI) double-staining cells early apoptosis rate; Western blot detection of 1, 2, 3, 4, 5 g · L -1 wart virus net intervention after 24 h H8 cell caspase-3 (Caspase-3), poly (ADP-ribose) polymerase (PARP) protein expression. Results: Compared with the blank group, the concentration of 2,3,4,5 g · L -1 wudujing had a significant concentration-time-dependent effect on the growth of H8 cells (P <0.05). After Hoechst 33342 staining 1, 2, 3, 4, 5 g · L ~ (-1) wart poisoning showed obvious chromatin condensation, 3,4,5 g · L ~ (-1) ) Wounded with bright green buds gradually increased under wart poisoning. The early apoptotic rate of cells was detected by flow cytometry after the wart poisoning of 1,2,3 g · L -1 for 24 h (23.73 ± 5.72)%, (63.9 ± 3.59)% and (71.53 ± 1.59)%, respectively. Compared with the blank group (6.53 ± 0.85)%, the early apoptosis rate was significantly increased (P <0.01) (P <0.05). The expression of Caspase-3 and its PARP protein in warts were significantly decreased (P <0.05), and the concentration of Caspase-3 was down-regulated. Conclusion: Wutun net can significantly inhibit the proliferation of cervical immortalized H8 cells and induce early apoptosis, which may be caused by the activation of Caspase-3-PARP pathway.