摘要
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。
Objective To study the effects of the cotreatment of cadmium chloride（CdCl2） and Smac over-expression on proliferation and apoptosis of hepatocellular carcinoma cells,SMMC-7721,and provide an experimental basis for the Smac-based tumor gene therapy and the development of cadmium as the antihepatoma drug.Methods SMMC-7721 were divided into five groups,which were control,pcDNA3.1＋,pcDNA3.1＋-hSmac,single CdCl2-treated and CdCl2 plus Smac groups respectively.The cells in control group had no transfection or CdCl2 treatment,while those in pcDNA3.1＋ and pcDNA3.1＋-hSmac groups were transfected with null vector pcDNA3.1＋ or pcDNA3.1＋-hSmac respectively through lipofectamine-mediated method,and those in CdCl2-treated groups were exposed to CdCl2 at the concentration of 10,20 and 30 μmol·L-1 respectively,and those in CdCl2 plus Smac groups were transfected with pcDNA3.1＋-hSmac firstly,and then treated with CdCl2 after transfection for 24 h.MTT assay was used to determine the effect of Smac over-expression and CdCl2 on proliferation,and both AO/EB fluorescence staining and FCM with Annexin Ⅴ and PI double-staining methods for apoptosis.Results The MTT results showed that compared with control group,except for the null-vector pcDNA3.1＋ group,the inhibitory rates in the rest groups were increased significantly（P〈0.01 or P〈0.001）.In single CdCl2-treated groups,the inhibitory rate was increased with the increasing of treatment doses,whereas,each Smac coupled group presented significantly higher inhibitory rate than the corresponding single CdCl2-treated group（P〈0.001）.AO/EB staining manifested that the cells in control group were in good condition,while those in pcDNA3.1＋-hSmac,30 μmol·L-1 CdCl2 treatment,and CdCl2 plus Smac groups appeared the morphological changes of apoptosis,especially in the last group.Furthermore,the results acquired by FCM showed that the apoptotic rate in pcDNA3.1＋-hSmac group was increased significantly when compared with control and pcDNA3.1＋ groups respectively（P〈0.001）.However,the apoptotic rate in CdCl2 pluse Smac group was the highest,which was significantly higher than that in single 30 μmol·L-1 CdCl2-treated group（P〈0.001）.Conclusion CdCl2 has cytotoxicity to SMMC-7721 cells.It could inhibit cellular proliferation in a dose-dependent manner,and induce apoptosis.Smac over-expression could inhibit cellular proliferation and slightly induce apoptosis independently.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2011年第4期571-576,F0002,共7页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金青年基金资助课题(30700672)
关键词
氯化镉
SMAC
凋亡
增殖
肝肿瘤
SMMC-7721
cadmium chloride； Smac； apoptosis； proliferation； liver neoplasms； SMMC-7721； cadmium chloride； Smac； apoptosis； proliferation； liver neoplasms； SMMC-7721； cadmium chloride； Smac； apoptosis； proliferation； liver neoplasms； SMMC-7721；
