Smac过表达增强氯化镉对人肝癌细胞SMMC-7721的增殖抑制和促凋亡作用

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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.
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