VEGF165质粒转染对骨髓内皮祖细胞增殖能力的影响

An in vitro study of PcDNA3.1(+)/VEGF165 transfection on endothelial progenitor cell derived from murine bone marrow

目的探讨脂质体介导pcDNA3.1(+)/VEGF165质粒转染骨髓血管内皮祖细胞(endothelial progenitor cells,EPCs)对EPCs增殖的影响。方法体外分离、培养EPCs、免疫组织化学及细胞流式仪鉴定EPCs,转染VEGF165后,采用反转录聚合酶链反应(RT-PCR)检测内皮祖细胞中VEGF165mRNA的表达,酶联免疫吸附法(ELISA)检测EPCs培养上清液中VEGF蛋白水平,噻唑蓝(MTT)检测VEGF165质粒转染后对EPCs的影响。结果EPCs表达CD34、CD133及KDR细胞表面标志,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。ELISA检测结果表明pcDNA3.1(+)/VEGF165质粒转染组VEGF165水平较其他两组高,MTT显示VEGF165质粒转染可促进EPCs增殖。结论EPCs可成功转染VEGF165基因,并促进EPCs增殖,为治疗勃起功能障碍提供实验依据。

Objective To investigate the effect of pcDNA3.1(+)/VEGF165 plasmid transfection on endothelial progenitor cells(EPCs)derived from murine bone marrow mediated by liposome.Methods EPCs were isolated from murine bone marrow cultured in vitro,identified by immunocytochemistry and fluorescence-activated cell sorting.After transfection,VEGF165 mRNA in EPCs was detected by reverse transcription polymerase chain reaction(RT-PCR),and VEGF protein in the culture supernatant of EPCs was detected by enzyme-linked immunosorbent assay(ELISA).MTT method was used to study the effect of transfection by pcDNA3.1(+)/VEGF165 plasmid on EPCs.Results EPCs expressed cell markers CD34,CD133,KDR.Gel electrophoresis showed that a 576bp 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 was higher than that in the other groups.VEGF165 plasmid transfer could stimulate EPCs proliferation.Conclusion EPCs can successfully transfect VEGF165 gene and promote the proliferation of EPCs,providing experimental basis for treatment of erectile dysfunction.