重组质粒pEGFP-ING4的构建及其对MCF-7细胞凋亡的影响

目的检测ING4(inhibitor of growth 4)对MCF-7细胞凋亡的影响。方法从人胎盘总RNA中克隆人ING4基因,构建其真核表达质粒pEGFP-ING4转染MCF-7细胞表达ING4后,流式细胞仪检测细胞凋亡率;荧光定量RT-PCR检测ING4、NF-κB、caspase-3、IL-8、Bcl-2及Bax基因的表达。结果构建的重组质粒转染可见目的蛋白融合表达,与对照相比MCF-7细胞凋亡率增高;ING4、caspase-3及IL-8基因的表达上调,NF-κB与Bcl-2/Bax基因的表达下调。结论成功从人胎盘组织克隆了ING4基因并构建其真核表达质粒在人MCF-7细胞中表达;ING4在人MCF-7细胞中能引起凋亡相关基因的改变并促进MCF-7细胞的凋亡,这为进一步研究ING4的抗肿瘤机制奠定了基础。

Construction of recombinant plasmid pEGFP-ING4 and its effects on apoptosis of MCF-7 cell line

We aim to observe the effects of human ING4 on apoptosis of MCF-7 cells.The gene of human ING4 was obtained from human placenta,and then cloned into eukaryotic expression vector pEGFP-C2 to construct recombinant protein expression vector pEGFP-ING4.Subsequently,the pEGFP-ING4 plasmid was transfected in human MCF-7 cell.The green fluorescence of expressed ING4 was observed under fluorescence microscope.After expression of ING4 protein,the cells apoptosis was detected using flow cytometry and the mRNA of ING4,NF-κB,caspase-3,IL-8,Bcl-2 and Bax were analyzed by QRT-PCR.Compared with the control,the apoptotic rate of MCF-7 cells was increased,the mRNA level of ING4,caspase-3 and IL-8 increased,but the mRNA levels of NF-κB and Bcl-2/Bax decreased.The results imply that the human ING4 may induce apoptosis of MCF-7 cells by increasing of apoptotic gene and decreasing of apoptotic suppressor gene in MCF-7 cells,which laid a basis for further research on its function and tumor suppress mechanisms.