核因子I-B高表达对肝L02细胞中蛋白磷酸酶2A-B55δ靶向调控研究  

Targeting regulation of protein phosphatase 2A-B55δ by nuclear factor-I/B in human liver L02 cells

作　　者：麦剑荣[1] 林育纯[1] 陈慧峰[1] 陈宇曦[1] 夏斌[1] 廖昆[1] 张玉静[1] 高艳芳[1] 林忠宁[1] 

机构地区：[1]广东省营养膳食与健康重点实验室中山大学公共卫生学院,广东广州510080

出　　处：《中国职业医学》2013年第3期181-185,共5页China Occupational Medicine

基　　金：国家自然科学基金资助项目(81172705,81072334,30771832);教育部科学技术研究重点项目(109126);教育部博士点基金(20090171110052);广东省自然科学基金重点项目(S2011020002769)

摘　　要：目的构建核因子I-B基因(NFIB)真核表达重组质粒,应用于其高表达调控肝细胞中蛋白磷酸酶2A(PP2A)的B亚基基因转录的功能学验证。方法逆转录聚合酶链反应(RT-PCR)获得人NFIB mRNA片段,克隆入真核绿色荧光蛋白表达载体(pEGFP-N1),构建pEGFP-NFIB重组质粒后,分别给予250、500、1 000 ng转染永生化人正常肝L02细胞建立NFIB高表达细胞模型(相应设为250、500和1 000 ng剂量组),对照组为250 ng pEGFP-N1转染的L02细胞,荧光定量聚合酶链反应检测NFIB mRNA,激光共聚焦和免疫印迹法(WB)检测核因子-1(NF-1)蛋白水平。电泳迁移率实验分析与NF-1相结合的DNA序列的特异性,荧光素酶报告基因检测PP2A-B55δ亚基基因(PPP2R2D)启动子区的转录活力,RT-PCR和WB检测细胞内PPP2R2D mRNA和PP2A-B55δ蛋白水平。结果双向测序证实pEGFP-NFIB重组表达质粒构建成功。与对照组比较,各剂量组L02细胞中NFIB mRNA相对水平增高(P<0.01),增强型绿色荧光蛋白、NFI-B蛋白表达水平均升高(P<0.05)。NF-1可与PPP2R2D基因启动子区(2R2Dp)-462G>A不同多态性序列结合,NFIB高表达使L02细胞中2R2Dp-462G启动子活力下降(P<0.05),PPP2R2D mRNA和PP2A-B55δ蛋白水平均升高(P<0.05)。结论成功构建人NFIB真核表达重组质粒,应用于人肝细胞中证实NF-1靶向PPP2R2D的功能学调控作用。Objective To construct the eukaryotic expression recombinant plasmid of nuclear factor-I/B（NFIB） gene and its functional identification of the transcriptional regulation of protein phosphatase 2A（PP2A） B subunits in human liver L02 cells.Methods A fragment of NFIB was amplified by reverse transcription-polymerase chain reaction（RT-PCR）,cloned into the pEGFP-N1 vector and the recombinant plasmid of pEGFP-NFIB was confirmed by sequencing.The pEGFP-NFIB was transiently transfected into immortal human hepatocyte L02 cell line which was divided into 250,500,1 000 ng dose group while the control group was transfected with 250 ng pEGFP-N1.The 4 groups were identified by real-time quantitative RT-PCR,laser confocal microscope and western blot.The transcription activity of the PP2A-B55δ subunit gene（PPP2R2D） promoter was detected by dual-luciferase reporter gene system.The expression of PP2A-B55δ mRNA and protein was detected by RT-PCR and western blot.Results The recombinant plasmid of pEGFP-NFIB identified by restriction enzyme digestion and RT-PCR reaction and confirmed successfully by two-way sequencing.Compared with control group,the each relative levels of NFIB mRNA in the 3 dose groups increased respectively（P ＆lt; 0.01）,and the expression of NF-1 and the green fluorescence were increased respectively（P ＆lt; 0.05）.The polymorphism in the PPP2R2D promoter region had an effect on the regulation of the translation factor.The-462G promoter activity of PPP2R2D,the expression of PPP2R2D mRNA and PP2A-B55δ protein were increased significantly in transfected cells（P ＆lt; 0.05）.Conclusion It was successful to construct and express the eukaryotic expression recombinant plasmid of NFIB in L02 cells.The results confirmed the foundation for research on the expression of the target gene PPP2R2D.

关 键 词：核因子-1 蛋白磷酸酶2A B55δ亚基 肝L02细胞 

分 类 号：R394.3[医药卫生—医学遗传学]