摘要
目的:探讨熊果酸(Ursolic Acid, UA)对人肝癌BEL-7404细胞的生长增殖抑制作用和诱导凋亡的机制。方法:以体外人肝癌BEL-7404细胞培养为基础,并设阴性对照组、实验组及阳性对照组进行实验,应用MTT法检测UA对肝癌BEL-7404细胞增殖的抑制作用,透射电子显微镜、Hoechst33258染色、流式细胞术检测细胞凋亡的发生,Western Blot检测凋亡相关蛋白procaspase-3、caspase-9及survivin表达,以探讨UA诱导肝癌BEL-7404细胞凋亡的可能机制。结果:①MTT结果表明UA对肝癌BEL-7404细胞具有增殖抑制作用,其中UA50μmol/L及DDP10μg/ml作用48小时的增殖抑制作用最佳,并作为本研究的实验条件进行实验;②透射电镜可见早期凋亡细胞膜外绒毛消失,内质网与胞膜融合,形成空泡。晚期凋亡细胞核内染色质浓缩,出现凋亡小体;③Hoechst33258染色示:凋亡细胞染色质浓染,呈现蓝白色荧光;④流式细胞术结果示:顺铂(ciplatin,DDP)组、UA组的凋亡率较阴性对照组高(F=3698.99,P<0.01);⑤Western Blot检测发现,凋亡相关蛋白procaspase-3、survivin表达降低,caspase-9表达增加。结论:UA对人肝癌BEL-7404细胞有显著的增殖抑制及诱导凋亡作用。机制可能为:UA作用肝癌BEL-7404细胞后,触发内源性凋亡信号传导通路,引起caspase-9的表达增加,进而作用于procaspase-3使其活化为活性caspase-3,同时,survivin蛋白被降解激活,进一步诱导细胞凋亡的发生。
Objective:To investigate the anti-tumor effect and apoptosis-inducing mechanism of UA on human hepatic carcinoma BEL-7404 cells. Methods:Based on the culture of human hepatic carcinoma BEL-7404 cells in vitro. Control group, UA group and DDP group were included. The effect of ursolic acid on growth of human hepatic carcinoma BEL-7404 cells was evaluated by MTT. To observe occurrence of apoptosis in the early, intermediate and terminal stage, the morphology, DNA fragmentation and phos- phatidylserine(PS) residues were detected by transmission electron microscopy, agarosegel electrophoresis and flow cytometry, respectively. Procaspase-3, caspase-9 and survivin proteins expression obtained by Western Blot analysis. Results:①MTT showed UA had a significant anti-proliferation effect on human hepatic carcinoma BEL-7404 cells,UA50μmol/L and DDP10μg/ml had the best anti-proliferation effect after 48h as the experiment condition.②Transmission electron microscopy showed early apoptosis cells had no villus, vacuole inside, the terminal stage apoptosis cells showed nucleus condensation and apoptotic bodies.③Hoechst33258 showed chromatin condensation with fluorescence after incubation by UA.④Result of flow cyto-metry showed apoptosis rate was increased after treatment with UA and DDP for 48h (F=3698.99, P<0.01).⑤Western Blot analysis indicated that expression level of procaspase-3 and survivin was descend, but caspase-9 increased. Conclusions:UA has the effect of anti-proliferation and inhibits apoptosis on BEL-7404 cells, the mechanism is that human hepatic carcinoma BEL-7404 cells were treated by UA can increase expression of caspase-9, but elevate the expression of procaspase-3, which can disjoint DNA, matrix proteins and framework proteins, at the same time, inhibit survivin activity, low down sensitivity to the factors of apoptotis, which intensify cell apoptosis.
引文
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