Elafin真核表达载体的构建及其对气道粘液高分泌的调节  

Construction of Eukaryotic expression vector pEGFP-N1-Elafin and its effects on mucus hypersecretion in human airway epithelial cells

作　　者：余红梅[1] 李琪[1] 尤列.皮尔曼 维克多.科罗索夫 周向东[1] 

机构地区：[1]重庆医科大学附属第二医院呼吸内科,重庆400010 [2]俄罗斯医学科学院远东呼吸生理与病理研究中心,布拉戈维申斯克675000

出　　处：《中国免疫学杂志》2011年第5期440-445,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(30770951);国家自然科学基金中俄国际合作项目(81011120108);中俄政府间合作项目(2009:13-01)

摘　　要：目的:构建天然内生多肽Elafin真核表达载体,探讨其对气道粘液高分泌的影响。方法:抽提Elafin行RT-PCR获取Elafin cDNA,双酶切后将片段装载到pMD18-T载体上。以pMD18-T-Elafin为模板行PCR反应,产物胶回收并双酶切后定向克隆至pEGFP-N1上,转化,筛选,双酶切鉴定重组质粒。将pEGFP-N1-Elafin转染正常人支气管上皮细胞HBE16,给予脂多糖(LPS)刺激,Western blot检测细胞内Elafin蛋白的相对含量,RT-PCR检测各组Elafin mRNA和粘蛋白(MUC)5AC mRNA表达水平,荧光素酶报告基因检测系统测定核转录因子-κB(NFκ-B)的活性;ELISA法分析各组细胞MUC5AC蛋白的相对含量。结果:成功构建Elafin真核表达载体,转染重组Elafin的HBE16细胞成功表达Elafin蛋白。LPS刺激可增强NF-κB的活性,该活性在转染重组Elafin后显著降低;MUC5AC蛋白含量及mRNA水平在LPS刺激后也显著升高,在转染重组Elafin后二者的表达水平明显降低。结论:Elafin真核表达载体成功构建,初步发现Elafin可通过降低NFκ-B的活性来下调MUC5AC的表达,为进一步深入研究其对气道粘液高分泌的调节机制奠定了基础。Objective：To construct eukaryotic expression vector pEGFP-N1-Elafin and explore the mechanisms of endogeny polypeptide elafin in regulating mucus hypersecretion in airway epithelial cells.Methods：Elafin gene was successfully cloned by RT-PCR reaction.Eukaryotic expression vector pEGFP-N1-Elafin was constructed,identificated by restriction enzyme reaction（EcoR Ⅰ and BamH Ⅰ） and DNA sequencing,and transfected into HBE16 cells.Cells were then stimulated by lipopolysaccharide（LPS）.After 24 h,the levels of Elafin protein and mRNA were detected by Western blot and RT-PCR respectively,the transcription activity of nuclear factor κB（NF-κB）were detected by luciferase reporter gene detection system,and the levels of MUC5AC protein and mRNA were detected by ELISA and RT-PCR.Results：The levels of Elafin protein and mRNA were obviously increased after transfection of recombinant Elafin into HBE16 cells,compared with LPS group or control group.There was an obvious increase of MUC5AC protein production and mRNA expression,with elevation of NF-κB activity,all significantly higher than control group.Transfected recombinant elafin reduced MUC5AC protein and mRNA level,inhibited NF-κB activity,compared with single LPS-stimulated group.Conclusion：Transfection of endogeny polypeptide Elafin may down-regulate MUC5AC overexpression by inhibition of NF-κB.

关 键 词：ELAFIN 粘蛋白 脂多糖 核转录因子-ΚB 

分 类 号：R363[医药卫生—病理学]