pcDNA3.1(+)/VEGF165质粒转染骨髓内皮祖细胞后VEGF165的表达  

Expression of vascular endothelial growth factor-165 in endothelial progenitor cells transfected with pcDNA3.1(+)/VEGF165 plasmid

作　　者：杨钰兴[1] 肖明朝[1] 苟欣[1] 邱明[1] 

机构地区：[1]重庆医科大学附属第一医院泌尿外科,重庆400016

出　　处：《重庆医科大学学报》2010年第5期663-666,共4页Journal of Chongqing Medical University

基　　金：重庆市自然科学基金计划项目(编号:2007BB5288)

摘　　要：目的:培养和鉴定大鼠内皮祖细胞(Endothelial progenitor cells,EPCs),观察pcDNA3.1(+)/VEGF165质粒转染大鼠骨髓内皮祖细胞后内皮细胞生长因子165(Vascular endot-helial growth fator-165,VEGF165)的表达。方法:体外培养内皮祖细胞,以免疫细胞化学法检测其表面抗原(CD34、CD133、KDR)的表达。通过pcDNA3.1(+)质粒转染VEGF165后,采用酶联免疫吸附法(ELISA)检测内皮祖细胞培养上清液中VEGF蛋白的水平,反转录聚合酶链反应(RT-PCR)检测内皮祖细胞中VEGF165 mRNA的表达。结果:免疫细胞化学检测表明培养的细胞为大鼠内皮祖细胞,PCR后凝胶电泳显示pcDNA3.1(+)/VEGF165质粒转染组在576bp处可见有明显条带,pcDNA3.1(+)空质粒转染组和未转染组可见微弱的电泳带位于500bp与600bp之间,约576bp大小。ELISA检测结果为pcDNA3.1(+)/VEGF165质粒转染组上清液中VEGF165水平为(175.8±10.7)pg/ml,pcDNA3.1(+)空质粒组为(10.5±1.6)pg/ml,未转染组为(9.3±1.3)pg/ml。结论:内皮祖细胞体外培养有微量VEGF165表达,应用pcDNA3.1(+)/VEGF165载体转染,可使SD大鼠内皮祖细胞高效表达VEGF165。Objective：To culture and identify ratendo the lialprogen it or cells,and too bserve the expression of recombin anthuman vascular endothelial growth fator-165（VEGF165）in endothelial progenitor cells transfected with pcDNA3.1（＋）/VEGF165 plasmid.Methods：Endothelial progenitor cells were cultured in vitro,and immunocytochemical staining was used to detect the expression of surface antigen（CD34,CD133,KDR）.After transfection of VEGF165,VEGF protein in the culture supernatant of endothelial progenitor cells was detected by enzyme-linked immunosorbent assay（ELISA）,and VEGF165 mRNA in endothelial progenitor cells was detected by reverse transcription polymerase chain reaction（RT-PCR）.Results：The cultured cells were identified to be endothelial progenitor cells by immunocytochemical analysis.Gel electrophoresis showed that a 576 bp product was amplified by RT-PCR in VEGF165-transfected cells,while a weak expression of VEGF165 was observed in mock-transfected and non-transfected cells.The protein level of VEGF165 in the supernatant of VEGF165-transfected,mock-transfected and non-transfected cells were 175.8±10.7 pg/ml,10.5±1.6 pg/ml and 9.3±1.3 pg/ml,respectively.Conclusion：There is small amount of VEGF165 expression in endothelial progenitor cells in vitro,and the endothelial progenitor cells transfected with pcDNA3.1（＋）/VEGF165 plasmid have an increased expression of VEGF165.

关 键 词：血管内皮生长因子 内皮祖细胞 转染 

分 类 号：R698.1[医药卫生—泌尿科学]