StAR基因高表达对DEHP致MCF-7细胞凋亡作用的影响  

Construction cells over-expressing the StAR gene and its apoptotic response to DEHP

作　　者：彭鹏[1,2] 王潮岗 徐新云 夏俊杰[1] 黄海燕[1] 王利[3] 黄奕钦[1] 杨晨 PENG Peng;WANG Chaogang;XU Xinyun;XIA Junjie;HUANG Haiyan;WANG Li;HUANG Yiqin;YANG Chen(Institue of Toxicology,Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,Guangdong;School of Life Science,Shenzhen University,Shenzhen 518060,Guangdong;School of Public Health,South China University, Hengyang 421001,Hunan,China)

机构地区：[1]深圳市疾病预防控制中心毒理研究所,广东深圳518055 [2]深圳大学生命与海洋科学学院,广东深圳518060 [3]南华大学公共卫生学院,湖南衡阳421001

出　　处：《癌变．畸变．突变》2018年第2期126-131,共6页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：深圳市科技研发基础研究项目(JCYJ20170306160553495)

摘　　要：目的:构建类固醇合成急性调节蛋白(St AR)基因高表达载体,建立St AR基因高表达细胞,研究St AR基因高表达对邻苯二甲酸二-(2-乙基己)酯(DEHP)毒性作用的影响。方法:根据Gen Bank提供的St AR基因c DNA序列设计引物,PCR扩增St AR基因并将其克隆到慢病毒载体中,构建St AR基因高表达MCF-7细胞,通过基因测序、荧光定量PCR和Western blot鉴定细胞。用不同剂量DEHP染毒MCF-7细胞和St AR基因高表达MCF-7细胞24 h,检测细胞凋亡相关基因Bax、Caspase-3、Caspase-8在m RNA和蛋白表达水平的变化。结果:基因测序证明构建的St AR基因高表达载体序列正确,荧光定量PCR检测St AR基因转染细胞比正常MCF-7细胞St AR m RNA表达水平升高591.9倍(P<0.01),Western blot实验结果显示,St AR基因转染细胞比正常MCF-7细胞St AR蛋白表达水平升高190%(P<0.01)。不同剂量DEHP染毒St AR基因高表达细胞后,荧光定量PCR结果显示,St AR基因高表达细胞中Bax、Caspase-3和Caspase-8 m RNA表达水平较对照组(0 mmol/L DEHP)显著升高,差异均有统计学意义(P<0.05或P<0.01)。Western blot实验显示,St AR基因高表达细胞中Bax、Caspase-3和Caspase-8蛋白表达水平比对照组显著升高,差异均有统计学意义(P<0.05或P<0.01)。结论:本实验成功构建了St AR基因高表达细胞,DEHP处理后St AR基因高表达细胞的凋亡相关基因表达水平较MCF-7细胞升高,提示St AR基因具有促进DEHP致细胞凋亡作用。OBJECTIVE:To construct cells with over-expression of the StAR gene and to study its apoptotic response to di-(2-ethylhexyl)phthalate(DEHP).METHODS:Primers were designed according to cDNA sequence of the StAR gene from GenBank.The gene was amplified using PCR and ligated into the lentiviral vector pLVX-CMV-ZSgreen-PGK-Puro.293FT cells were transfected with the recombinant vector and then MCF-7 cells were transfected.Cells with over-expression of the StAR gene were identified by gene sequencing,real-time quantitative PCR and western blot.Then the StAR gene over-expression cells and MCF-7 cells were treated with various doses of DEHP for 24 h to detect expression of apoptosis genes including Bax,Caspase-3 and Caspase-8.RESULTS:The sequence contained in the recombinant vector was exactly the same as the StAR gene from GenBank.StAR gene expression level in the transfected MCF-7 cells was 591.9 times higher than that of control MCF-7 cells.StAR protein level in the transfected MCF-7 cells was 190%higher than that of control MCF-7 cells.After DEHP treatment of the transfected and control MCF-7 cells,qPCR results show that mRNA levels of Bax,Caspase-3 and Caspase-8 were significantly higher in the transfected MCF-7 cells.Additionally,mRNA levels of Bax,Caspase-3 and Caspase-8 increased significantly(P<0.05 or P<0.01)in the transected compared with the similarly treated non-transfected MCF-7 cells.Western blot results show that the protein expression levels of Bax,Caspase-3 and Caspase-8 also increased significantly in the transfected MCF-7 cells.Protein expression levels of Bax,Caspase-3 and Caspase-8 were also increased significantly(P<0.05 or P<0.01)except in the 0.2 mmol/L treatment group.CONCLUSION:The StAR gene over-expression cells were successfully constructed.These cells responded to DEHP treatments by showing significant increase in apoptotic gene expression.These findings indicate that StAR gene promoted DEHP induced-apoptosis.

关 键 词：邻苯二甲酸二-(2-乙基己)酯 类固醇合成急性调节蛋白 基因高表达 凋亡基因 

分 类 号：R394.6[医药卫生—医学遗传学]