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目的研究硒-甲基硒代半胱氨酸(MSC)对肝癌SMMC-7721细胞株抑制及凋亡的作用,探讨其分子机制。方法试验分空白对照组和MSC组(细胞培养体系中加入MSC)。MTT法观察细胞增殖;流式细胞术检测细胞周期及凋亡率;透射电镜观察细胞超微结构的改变;琼脂糖凝胶电泳法观察DNA的“梯状”条带;Western blot方法检测凋亡诱导因子(AIF)蛋白表达。结果随着MSC浓度和作用时间增加,细胞抑制率上升。肝癌细胞株SMMC-7721在MSC浓度为25、50、100、200μmol/L下,24h细胞抑制率分别为(14.44±3.83)%、(21.15±0.61)%、(50.64±2.40)%、(64.25±0.87)%,48h细胞抑制率分别为(35.97±2.08)%、(57.41±2.61)%、(74.71±1.59)%、(91.68±1.33)%(P<0.05)。MSC组细胞周期出现S期阻滞,并出现凋亡峰,50、100μmol/L浓度的MSC干预48h后,肝癌SMMC-7721细胞株凋亡率分别为28.46%、50.73%。MSC组细胞呈凋亡表现,核固缩、碎裂,染色质浓缩,边集。MSC 50μmol/L或100μmol/L组培养48h后,DNA琼脂糖凝胶电泳可见典型的梯状条带;而空白对照组细胞DNA在电泳上未见梯状条带。经MSC干预后,AIF蛋白表达呈浓度依赖性增多(P<0.05)。结论 MSC对肝癌MMC-7721细胞株有抑制增殖、促进凋亡的作用。其机制可能通过调控AIF表达激活非Caspases依赖凋亡通路实现的。
Objective To study the effect of selenium-methylselenocysteine (MSC) on the inhibition and apoptosis of hepatocellular carcinoma cell line SMMC-7721 and its molecular mechanism. Methods The experiment was divided into blank control group and MSC group (added MSC into cell culture system). MTT assay was used to observe the cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis rate. Transmission electron microscopy was used to observe the changes of cell ultrastructure. DNA ladder was observed by agarose gel electrophoresis. Apoptosis inducing factor (AIF) protein expression. Results As the concentration of MSCs and the action time increased, the cell inhibition rate increased. The inhibitory rates of hepatoma cell line SMMC-7721 at 24h, 50,100 and 200μmol / L were (14.44 ± 3.83)%, (21.15 ± 0.61)%, (50.64 ± 2.40)% and ± 0.87%, 48h respectively. The inhibitory rates were (35.97 ± 2.08)%, (57.41 ± 2.61)%, (74.71 ± 1.59)% and (91.68 ± 1.33)%, respectively. The cell cycle arrest of S phase and the appearance of apoptotic peak appeared in MSC group. The apoptotic rate of SMMC-7721 cells treated with 50 and 100 μmol / L of MSCs for 48 h were 28.46% and 50.73%, respectively. MSC cells showed apoptosis, nuclear pyknosis, fragmentation, chromatin condensation, edge set. After cultured in MSC 50μmol / L or 100μmol / L for 48h, typical ladder-like bands were observed by DNA agarose gel electrophoresis. However, no ladder-like bands were observed in the blank control cells. After intervention by MSC, AIF protein expression increased in a concentration-dependent manner (P <0.05). Conclusion MSC can inhibit proliferation and promote apoptosis of hepatocellular carcinoma cell line MMC-7721. The mechanism may be through the regulation of AIF expression activation of non-Caspases dependent apoptosis pathway.