An In Vitro Study Of PCDNA3.1(+)/VEGF₁₆₅ Transfection On Endothelial Progenitor Cell Derived From Murine Bone Marrow

Objective To construct the pcDNA3.1(+)/VEGF165 eukaryotic expression vector and to investigate the study of endothelial progenitor transfected with pcDNA3.1(+)/VEGF165 plasmid in vitro.Methods VEGF165 gene was cloned to pCR2.1-TOPO vector, and then cloned into the vector pcDNA3.1(+) after sequencing. EPCs were isolated from murine bone marrow cultured in vitro,identified by immunocytochemistry.There were three groups, EPCs transfected with pcDNA3.1(+)/VEGF165,EPCs transfected with pcDNA3.1(+) and EPCs without transfection.After transfection, 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). MTT method was used to study the effect of transfection by pcDNA3.1(+)/VEGF165 plasmid on EPCs.Results pcDNA3.1(+)/VEGF165 was successfully constructed, and confirmed by gene sequencing, enzyme digestion and PCR identification. EPCs expressed cell markers CD34, CD133, KDR. The protein level of VEGF165 in the supernatant of VEGF165-transfected were more than the other groups(P<0.05).Gel electrophoresis showed that VEGF165 mRNA was detected in endothelial progenitor cells. VEGF165 plasmid transfer could stimulate EPCs proliferation.Conclusion pcDNA3.1(+)/VEGF165 was successfully constructed and VEGF165 gene could be successfully transfected into EPCs, 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 and VEGF produced by gene thansfer can stimulate EPCs proliferation.