PDCD4在Hela细胞中过表达致抗凋亡蛋白Bcl-2表达下调机制探讨  

PDCD4 overexpression suppresses the levels of anti-apoptosis gene Bcl-2 in Hela cells

作　　者：郑伟[1] 王聪洋 姜艳[2] 杨大群[2] 高广东[2] 孙中满 李珊[1,2] 

机构地区：[1]湖北医药学院附属东风医院综合医疗科,湖北十堰442000 [2]湖北医药学院生物化学教研室,湖北十堰442000

出　　处：《现代肿瘤医学》2016年第17期2690-2694,共5页Journal of Modern Oncology

基　　金：湖北省自然科学基金面上项目青年基金(编号:2014CFB647);湖北省教育厅科学技术研究计划优秀中青年人才项目(编号:Q20142103);湖北医药学院研究生启动金(编号:2013QDJZR05)

摘　　要：目的:探讨程序性细胞死亡因子4(PDCD4)的抑癌机制。方法:以含有全长PDCD4的c DNA为模板,扩增PDCD4全长基因,融合PDCD4-GFP融合基因真核表达载体,将其转染Hela细胞,采用激光共聚焦显微镜观察融合蛋白在细胞中的表达,以荧光定量PCR和Western-blot检测PDCD4在细胞内的表达以及抗凋亡基因Bcl-2的表达情况。结果:经酶切图谱分析、PCR扩增及DNA测序证实融合基因真核表达载体构建正确;荧光显微镜可观察到细胞内PDCD4-GFP融合蛋白的表达;RT-PCR证实经PDCD4基因转染的细胞内PDCD4 mRNA表达增高。PDCD4的过表达能够导致Bcl-2基因表达下调。结论:PDCD4基因表达于宫颈癌细胞核和胞浆内,并且PDCD4能够抑制Bcl-2基因的表达水平。Objective：To understand the function of anti-tumor gene-programmed cell death 4（ PDCD4）,and provide a useful tool for the prevention of human cervical tumor.Methods：The full length of PDCD4 was amplified by polymerase chain reaction（ PCR）,using c DNA as a template.The recombinant plasmid PDCD4-p EGFP-N1 was confirmed by restricted endonuclease mapping,PCR amplification and DNA sequencing.Hela cells were transfected with PDCD4-p EGFP-N1 to investigate the expressions of PDCD4.Fluorescence microscopy was employed to observe the expression of PDCD4-GFP fusion protein,Real-time PCR was used to analyze the expression of PDCD4 gene in Hela cells,and protein levels of Bcl-2.Results：The expression vector PDCD4-p EGFP-N1 was successfully constructed and resulted in simultaneously cellular expression of both PDCD4 and GFP when transfected into Hela cells.Fluorescence microscopy confirmed the GFP expression in the cells and RT-PCR revealed that the expression of PDCD4 mRNA was increased in PDCD4 gene transfected cells,PDCD4 overexpression lead to reduced level of Bcl-2 mRNA.Conclusion：It is demonstrated the location of PDCD4 and simultaneously express PDCD4 in Hela cells which lead to decrease of Bcl-2 expression,which provide the evidence that how PDCD4 function as a tumor suppress gene.

关 键 词：PDCD4基因 BCL-2 HELA细胞 

分 类 号：R730.3[医药卫生—肿瘤]