Transfection of Adenovirus-mediated Vascular Endothelial Growth Factor into Umbilical Cord Mesenchymal Stem Cells in Vitro

Objective To investigate the feasibility of transfection of adenovirus-mediated vector vascular endothelial growth factor( VEGF) into umbilical cord mesenchymal stem cells( UCMSCs) and the effect of VEGF transfection on UCMSCs function. Methods VEGF165- EGFP genetic recombination of adenovirus vectors were constructed,and UCMSCs were isolated and cultured,and then adenovirus vector-enhanced green fluorescent protein( EGFP) transfected UCMSCs. The transfection effect was observed under inverted fluorescence microscope,and transfection effectiveness was detected by flow cytometer so to confirm the best multiplicity of infection( MOI). Cells were divided into VEGF-EGFP transfection group,EGFP group and control group,and the cells were transfected and collected based on the best MOI. Expressions of VEGF and protein kinase B( PKB also named Akt) signaling protein were detected by Western blotting; the expression of VEGF protein was detected by enzyme linked immunosorbent assay( ELISA). The proliferation effect of transfected UCMSCs was evaluated with CCK 8 method. Results Adenovirus-mediated VEGF165-EGFP gene successfully transfected UCMSCs,and VEGF gene was transcribed and expressed in cells,and then was secreted out of cells. Levels of VEGF and Akt transfection secretion in VEGF-EGFP group 48 hours after transfection were significantly higher than those in EGFP group and control group; the expression and secretion of VEGF could be detected in VEGF-EGFP transfection group by ELISA 48 hours after transfection,and the peak appeared 4thd after transfection,at the same time the expressions in VEGF group were significantly higher than those in EGFP group and control group( P 0. 01),and then the levels gradually decreased,but can maintain stable expression for several days. Results of CCK 8 method showed that VEGF gene transfection had no significant effect on the UCMSCs proliferation. Conclusion Adenovirus-mediated VEGF-EGFP gene may successfully transfect UCMSCs,maintain UCMSCs original biological characteristics,and significantly and efficiently express VEGF,which can provide theoretical basis for the feasibility of VEGF gene combined with UCMSCs in treatment of diabetic lower limb blood circulation reconstruction.