DOTAP liposome-mediated transfection of human adipose-derived stemcells with pIRES2-EGFP-VEGF plasmid and target gene expression

Objective:To explore liposome-mediated transfection of human adipose-derived stem cells (hASCs) with vascular endothelial growth factor(VEGF) gene and to investigate the expression of VEGF after transfection.Method:Lipoaspirate was digested using collagenase.Cell pellet was harvested and subcultured to passage 4.Phenotype was detected with flow cytometry and multilineage differentiation was induced for the identification of hASCs.hASCs was transfected with pIRES2-EGFP-VEGF plasmid using DOTAP liposome.The intracellular expression of VEGF was detected by immunofluorescent staining and the VEGF concentration in supernatant was analyzed by ELISA.Result:1 ml lipoaspirate yielded(4.38±0.21)×10⁵ cells.hASCs on passage 4 showed high expression of CD90(81.49%) and low expression of CD19(6.37%),CD31(14.91%),CD34(17.56%) and CD45(15.39%).GFP and VEGF were observed in transfected hASCs.The transfection efficiency was(43.69±18.53)%.Untransfected hASCs did not express GFP but low level of VEGF.The optical density of VEGF intransfected hASCs is 2.13 fold of untransfected hASCs.The VEGF concentration in supernatant of transfected hASCs significantly increased over time and exhibit statistic differences compared with untransfected hASCs(P<0.05).Conclusion:hASCs were successfully transfected with pIRES2-EGFP-VEGF plasmid using DOTAP liposome.The post-transfection expression and secretion of VEGF remarkably increased.