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目的:通过研究雷公藤内酯醇(TP)对COC1/DDP体外活性的影响,初步探讨雷公藤内酯醇促进COC1/DDP细胞凋亡的机制,为TP成为治疗晚期或耐顺铂卵巢癌药物提供实验依据。方法:采用MTT法检测顺铂(DDP)、TP及两者联用对COC1/DDP的生长抑制作用;用透射电镜观察TP作用24h后细胞超微结构的变化;采用流式细胞术分析不同浓度TP对COC1/DDP细胞周期和细胞凋亡的影响;用DNA电泳分析用药后细胞基因组DNA断裂状况;以免疫组化分析TP对Caspase-7蛋白表达的影响。结果:DDP在24h、48h和72h三个时间段对COC1/DDP细胞的半数抑制量(50%concentration of inhibi-tion,IC50)分别为5.567μg/ml、2.866μg/ml和1.161μg/ml,其对COC1/DDP细胞增殖表现出浓度-时间依赖性的抑制作用(P<0.05);TP可抑制COC1/DDP细胞增殖,其抑制率呈浓度-时间依赖性(P<0.05);TP作用细胞24h后,电镜下可见凋亡小体形成;流式细胞术分析表明,各浓度TP组G1期细胞明显升高,细胞凋亡率呈浓度依赖性(P<0.05);DNA电泳分析可见细胞凋亡特有的DNA断裂形成的“梯形”条带;免疫组化表明Caspase-7参与了TP诱导COC1/DDP细胞凋亡过程。结论:TP对COC1/DDP具有明显的杀伤和促凋亡作用,凋亡机制与Caspase-7蛋白表达上调有关。
OBJECTIVE: To investigate the mechanism of triptolide on COC1 / DDP cell apoptosis by studying the effect of triptolide (TP) on the activity of COC1 / DDP in vitro and to provide experimental evidence for TP as a drug for the treatment of advanced or cisplatin-resistant ovarian cancer in accordance with. Methods: MTT assay was used to detect the growth inhibition of COC1 / DDP induced by cisplatin (DDP) and TP. The ultrastructural changes of COC1 / DDP cells were observed by transmission electron microscopy. Flow cytometry TP on the cell cycle and apoptosis of COC1 / DDP cells. DNA fragmentation was analyzed by DNA electrophoresis. The effect of TP on Caspase-7 protein expression was analyzed by immunohistochemistry. RESULTS: The IC50 values of DDP for COC1 / DDP cells in three time periods of 24h, 48h and 72h were 5.567μg / ml, 2.866μg / ml and 1.161μg / ml respectively, The proliferation of COC1 / DDP cells showed a concentration-time dependent inhibition (P <0.05). TP inhibited the proliferation of COC1 / DDP cells in a time-and concentration-dependent manner (P <0.05) The apoptotic bodies were observed under electron microscope after 24 hours. The results of flow cytometry showed that the cells in G1 phase were significantly increased and the cell apoptosis rate was increased in a concentration-dependent manner (P <0.05) Caspase-7 was involved in TP-induced apoptosis in COC1 / DDP cells by immunohistochemistry. CONCLUSION: TP has obvious anti-apoptosis and apoptosis effects on COC1 / DDP. The mechanism of apoptosis is related to the up-regulation of Caspase-7 protein expression.