Co-transfection Of Adeno-associated Virus-mediated Human Vascular Endothelial Growth Factor165 And Stromal Cell Derived Factor-1 Into Endothelial Progenitor Cells Of Mouse

Objective Sepsis is a severe clinical syndrome,It is the main cause of multiple organ failure and death in ICU.The damage and activation of endothelial cells(ECs)play an important role in the pathogenesis of sepsis,we investigated the feasibility of human vascular endothelial growth factor 165(hVEGF165)and stromal cell derived factor-1(SDF-1)gene therapy for sepsis.Methods The recombinant plasmid of CAG-VEGF165-T2A-mcherry-WPRE and pHBAAV-tfeb-MCS-3flag-T2A-ZsGreen was constructed by gene recombination technology,identified by enzyme digestion and sequencing identification,and then transfer to the HEK293 cell.After isolation and culture of vascular endothelial progenitor cells,the expression of VIII factor,KDR,CD34 and CD31 were analyzed by immunohistochemistry,the expression of CD34 in mononuclear cells and adherent cells cultured for 7 days were detected by flow cytometry,and the ability of cells to uptake Dil-Ac-LDL and to bind FITC-UEA-1 were identified.The protein hVEGF165 and SDF-1 were detected by Western Blot,and explored the influence to the proliferation of EPCs by MTT.Results It was confirmed by enzyme digestion and gene sequencing that the target gene sequence was basically consistent.Western Blot showed that the protein hVEGF165 and SDF-1 were well over expressed,and the bioactive AAV-hVEGF165 and AAV-SDF-1 were successfully constructed.Immunohistochemistry showed that VIII,KDR,CD34 and CD31 were successfully expressed in the vascular endothelial cells.Flow cytometry showed that CD34+ was almost not expressed in the blank group.A small number of cells in the newly isolated mononuclear cell group expressed CD34+ and a large number of cells expressed CD34+ after 7 days of culture.Fluorescence microscopy showed that most of the primary cultured EPCs were double positive and accorded with the characteristics of EPCs.The hVEGF165 and SDF-1 could promote the proliferation of vascular EPCs,the expression of the protein hVEGF165 and SDF-1 were manifested in vascular EPCs by Western Blot,and its in vascular EPCs co-transfected by AAV-hVEGF165 and AAV-SDF-1 was obviously more than that of the monogenic transfected cell.Conclusion : Co-transfection of AAV-hVEGF165 and AAV-SDF-1 can significantly increase the expression of the protein hVEGF165 and SDF-1 in endothelial progenitor cells.