雌激素受体α真核表达载体的构建及在大鼠骨髓基质干细胞中的表达  

Construction of a recombinant eukaryotic expression vector of ERα gene and its expression in rat marrow mesenchymal stem cells

作　　者：祝强[1] 王婧[2] 董隽[1] 史立新[1] 张旭[1] 洪宝发[1] 

机构地区：[1]解放军总医院泌尿外科,北京100851 [2]解放军总医院泌尿外科口腔科,北京100851

出　　处：《医学研究生学报》2014年第1期34-37,共4页Journal of Medical Postgraduates

摘　　要：目的雌激素在很多靶器官中发挥重要生物调节作用,主要通过雌激素受体(estrogen receptor,ER)来介导。构建ERα真核表达载体并于大鼠骨髓基质干细胞(rat marrow mesenchymal stem cells,rBMSCs)中表达,为后续研究ERα的功能奠定良好的基础。方法采用RT-PCR技术从SD大鼠子宫及双侧附件中提取并扩增ERα,酶切后,克隆至真核表达载体pcDNA3.1(+);重组载体经PCR、酶切、测序鉴定正确后瞬时转染rBMSCs,采用RT-PCR和Western blotting分析ERα在细胞中的表达,并采用Realtime-PCR和MTT的方法比较ERα高表达后对细胞生长周期的影响。结果成功构建了重组质粒pcDNA-ERα,RT-PCR和Western blotting显示该质粒能在rBMSCs中高效表达,Realtime-PCR和MTT比较分析表明,ERα的高表达对细胞的生长产生一定的抑制作用。结论成功构建ERα真核表达载体,该载体在rBMSCs中成功转录与表达,外源性ERα基因转染能使rBMSCs的增殖受抑,可能与ERα的核内高表达相关。Objective Estrogen 17-β estrodiol plays a very important part in a wide variety of tissues and cells, including those of the brain, breast, cardiovascular system and uterus by binding to estrogen receptors （ERs） to mediate various hormonal effects. This study was to construct a eukaryotic expression vector carrying estrogen receptor-a （ERoL） and study its transient expres- sion in rat marrow mesenchymal stem cells （rBMSCs）. Methods We amplified the ERa obtained from SD rats by RT-PCR and cor-rectly inserted it into the corresponding sites of the eukaryotic expression vector pcDNA3.1 （＋） following restriction endonuelease diges-tion. We transfected the recombinant plasmid into rBMSCs, examined the expression of ERa in the cells by RT-PCR and Western blot- ting, and determined the effect of the ERot gene transfection on the proliferation of rBMSCs by and real time PCR. Results The recombinant plasmid pcDNA-ERawas successfully established and highly expressed in rBMSCs. The results of real time PCR and MTT showed that the high expression of ERa inhibited the proliferation of rBMSCs. Condusion The eukaryotie expression vector carrying ERa was successfully constructed and transiently expressed in rBMSCs. The transfection of the ERa gene suppresses the prolif- eration of rBMSCs, which may be associated with the over-expression of ERa in cell nuclei.

关 键 词：雌激素受体理 基因表达 大鼠骨髓基质干细胞 转染 鉴定 

分 类 号：Q78[生物学—分子生物学]