| Objective: To Construct of vascular endothelial growth factor 165(VEGF165) recombinant adenovirus (Ad-VEGF165). After infection with the recombinant Ad-VEGF165,EPCs were examined for the expression of VEGF,MTT assay was employed to assess the growth character.Methods: The recombinant adenoviral vector carrying the VEGF165 gene was constructed by pAdEasy system.pAdEasy-VEGF165 transfected into HEK293A cells to package the adenovirus,followed by identification by means of GFP.PCR and gene sequencing identify the recombinant.EPCs were isolated from rat bone marrow by Ficoll and cultured with EBM-2MV medium cultivation.MTT assay were used to draw growth curve of cells.After infection with the recombinant Ad-VEGF165,EPCs were examined with a fluorescent microscopy and ELISA for their expression of VEGF,MTT assay was employed to assess the growth character,and Laser scanning confocal microscope for transduction efficiency.Results:Ad-VEGF165 was constructed and successfully cultivated a bone marrow-derived endothelial progenitor cells.Appropriate ratio of transfection had no significant effect on the growth of EPCs.When the MOI was 50,the transfection efficiency was 57.4 % .After transfection,VEGF protein was highly secretioned by EPCs.Conclusion:Ad-VEGF165 was constructed successfully,and Ad-VEGF165 could successfully transfected EPCs.Appropriate ratio of EPCs transfection had certain energetic impact on growth and activity,and EPCs transfected by Ad-VEGF165 could highly expressed VEGF protein. |
