Construction of eukaryotic expression plasmid of pcDNA3.1/VEGF-B-EGFP and its expression in bone marrow mesenchymal stem cells

Objective To detect the expression of vascular endothelial cell growth factor(VEGF)-B in human bone marrow mesenchymal stem cells(MSCs).Methods The recombinant plasmids of pcDNA 3.1/VEGF-B-EGFP were obtained by polymerase chain reaction(PCR) amplification,splicing,and insertion of complementary deoxyribonucleic acid(cDNA) fragments of VEGF-B and EGFP.The target gene was transferred to the third cultured MSCs(pcDNA 3.1/VEGF-B-EGFP transferred group) by using of cationic liposome mediated gene transfection technique.EGFP expression was detected with fluorescence microscopy,and the transfection rate was determined with flow cytometry.Reverse transcriptase PCR(RT-PCR),Western blot and other techniques were used to detect the expression of VEGF-B gene.Results In pcDNA 3.1/VEGF-B-EGFP transferred group,bright green fluorescence was observed with fluorescence microscopy 24 hours after transfection in MSCs,the rate of transfection analyzed with flow cytometry was 15%.RT-PCR results showed that the genome of VEGF-B transferred MSCs contained a 590bp specific fragment of VEGF-B gene.Western blot results proved VEGF-B protein expression in MSCs transferred by VEGF-B.Conclusion The generation of eukaryotic expression plasmid carrying VEGF-B gene and its expression in MSCs provide a sound basis for the combination of VEGF-B gene therapy and autologous MSCs transplantation in ischemic heart disease.