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目的:研究Smac过表达联合氯化镉(CdCl2)对人肝癌细胞SMMC-7721增殖和凋亡的影响,为以Smac为基础的肿瘤基因治疗以及镉作为抗肝癌药物的开发提供实验依据。方法:实验共设立5组,分别为对照组、pcDNA3.1+组、pcDNA3.1+-hSmac组、CdCl2组以及CdCl2联合Smac组。对照组细胞不进行细胞转染、不染毒;pcDNA3.1+和pcDNA3.1+-hSmac组细胞采用脂质体介导的方法分别转染空载体pcDNA3.1+和携带Smac基因的重组质粒pcDNA3.1+-hSmac;CdCl2组给予不同浓度(10、20和30μmol.L-1)CdCl2;CdCl2联合Smac组细胞首先将pcDNA3.1+-hSmac转染入SMMC-7721,然后给予CdCl2处理。采用MTT法检测Smac过表达联合CdCl2对细胞增殖的影响,AO/EB法和AnnexinⅤ/PI双荧光标记流式细胞术(FCM)检测细胞凋亡。结果:MTT结果显示,与对照组比较,除空载体pcDNA3.1+组无统计学意义外,其余各组细胞A490值均降低,抑制率均明显增加,差异具有统计学意义(P<0.01或P<0.001);单纯CdCl2组细胞抑制率随剂量增加而显著上升;分别与相应的单纯CdCl2组和pcDNA3.1+-hSmac组比较,CdCl2联合Smac组细胞的抑制率明显升高(P<0.001)。AO/EB染色荧光显微镜下观察显示,对照组细胞状态良好,而pcDNA3.1+-hSmac组、30μmol.L-1 CdCl2组及CdCl2联合Smac组细胞出现凋亡形态学改变,且后者最为明显。FCM结果显示,pcDNA3.1+-hSmac组细胞凋亡率增加,分别与对照组和pcDNA3.1+组比较,差异均具有统计学意义(P<0.001)。CdCl2联合Smac组细胞凋亡率最高,显著高于单纯30μmol.L-1 CdCl2组(P<0.001)。结论:CdCl2对SMMC-7721细胞具有毒性,可抑制细胞增殖,呈现剂量-效应关系,可诱导细胞凋亡。Smac过表达可抑制SMMC-7721细胞增殖,诱导细胞凋亡,并可增强CdCl2对SMMC-7721细胞的增殖抑制和促凋亡作用。
AIM: To investigate the effects of Smac overexpression combined with cadmium chloride (CdCl2) on the proliferation and apoptosis of human hepatocellular carcinoma cell line SMMC-7721, to provide experimental evidence for Smac-based tumor gene therapy and cadmium as an anti-hepatoma drug. Methods: A total of 5 groups were established, which were control group, pcDNA3.1 + group, pcDNA3.1 + -hSmac group, CdCl2 group and CdCl2 combined with Smac group. The cells in the control group were not transfected with cells, and were not infected. The pcDNA3.1 + and pcDNA3.1 + -hSmac cells were transfected with pcDNA3.1 + vector and Smac gene by liposome respectively pcDNA3.1 + -hSmac; CdCl2 groups were given different concentrations (10, 20 and 30μmol.L-1) of CdCl2; CdCl2 and Smac group cells transfected with pcDNA3.1 + -hSmac into SMMC-7721, then treated with CdCl2. The effect of Smac overexpression combined with CdCl2 on cell proliferation was detected by MTT assay. Apoptosis was detected by AO / EB and AnnexinⅤ / PI double fluorescent labeling flow cytometry (FCM). Results: Compared with the control group, the results of MTT assay showed that the A490 values of all the other groups were significantly lower than those of the control group except for the pcDNA3.1 + empty vector group, and the inhibition rates were significantly increased (P <0.01 or (P <0.001). The inhibitory rate of cells in CdCl2 group increased significantly with the increase of dose. Compared with CdCl2 group and pcDNA3.1 + -hSmac group, the inhibition rate of CdCl2 group and Smac group increased significantly (P <0.001) ). AO / EB staining under fluorescence microscope showed that the control group cells in good condition, while the pcDNA3.1 + -hSmac group, 30μmol.L-1 CdCl2 group and CdCl2 combined with Smac group apoptosis morphological changes, and the latter the most obvious . FCM results showed that the apoptosis rate of pcDNA3.1 + -hSmac group increased, respectively, compared with the control group and pcDNA3.1 + group, the difference was statistically significant (P <0.001). The apoptosis rate of CdCl2 combined with Smac group was the highest, significantly higher than that of 30μmol.L-1 CdCl2 group (P <0.001). Conclusion: CdCl2 is toxic to SMMC-7721 cells and inhibits cell proliferation, showing dose-effect relationship and inducing apoptosis. Smac overexpression can inhibit the proliferation of SMMC-7721 cells, induce apoptosis, and enhance the proliferation inhibition and apoptosis of SMMC-7721 cells induced by CdCl2.