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目的探讨反义survivin-脂质体复合物(survivin-ASODN)对肝癌细胞系HepG2的生长抑制作用及其机制,为肝癌及survivin阳性肿瘤的治疗提供理论依据。方法用脂质体介导survivin-ASODN转染肝癌细胞系HepG2细胞;通过RT-PCR和Westernblot检测survivin表达水平;以MTT法测量细胞生长情况;以流式细胞术测定caspase-3活性及凋亡率,电镜观察细胞形态学变化,并应用流式细胞仪同步分析survivin-ASODN对肝癌细胞系HepG2增殖周期的影响。结果surviving-ASODN可有效下调survivin表达水平,并呈剂量依赖关系,其IC50值为250nmol/L,最大效应浓度为600nmol/L;可抑制细胞生长,激活caspase-3活性,诱导细胞凋亡,使其出现凋亡形态学变化,如胞浆空泡变性、核浓缩和核碎裂。细胞周期的同步分析显示,surviving-ASODN对HepG2细胞增殖周期有明显影响,HepG2先出现细胞周期阻滞,紧接着出现细胞凋亡;低浓度处理后,可将细胞先后阻滞在S期和G2/M期;高浓度时,S期阻滞被加强,可快速诱导细胞凋亡,并且,细胞凋亡率随着药物浓度的增加和作用时间的延长明显上升。结论surviving-ASODN对肝癌细胞有强的生长抑制效应,其机制是与阻断细胞周期及诱导细胞凋亡有关。
Objective To investigate the inhibitory effect of survivin-ASODN on the growth of hepatocellular carcinoma cell line HepG2 and its mechanism, and to provide a theoretical basis for the treatment of hepatocellular carcinoma and survivin-positive tumors. Methods Liposome-mediated transfection of survivin-ASODN into hepatoma cell line HepG2 was performed. The expression of survivin was detected by RT-PCR and Western blotting. The cell growth was measured by MTT assay. The activity of caspase-3 and apoptosis The morphological changes of cells were observed by electron microscopy. The effects of survivin-ASODN on the proliferation of HepG2 cells were analyzed by flow cytometry. Results Survivin-ASODN could down-regulate the expression of survivin in a dose-dependent manner. The IC50 value was 250 nmol / L and the maximum concentration was 600 nmol / L. Survivin-ASODN could inhibit cell growth, activate caspase-3 activity and induce apoptosis The apoptotic morphological changes, such as cytoplasmic vacuolar degeneration, nuclear condensation and nuclear fragmentation. Synchronous analysis of cell cycle showed that surviving-ASODN had a significant effect on the proliferation cycle of HepG2 cells. HepG2 first appeared cell cycle arrest, followed by apoptosis. After low concentration treatment, cells could be arrested in S phase and G2 / M phase. At high concentration, S phase arrest was enhanced and apoptosis was rapidly induced. Moreover, the apoptosis rate increased significantly with the increase of drug concentration and the prolongation of action time. Conclusion Survivin-ASODN has a strong growth-inhibiting effect on hepatocellular carcinoma cells. Its mechanism is related to blocking the cell cycle and inducing apoptosis.