Abstract:

Objective To construct the eukaryotic expression plasmid pIRES2-EGFP-hVEGF165 of  human vascular endothelial growth factor 165(hVEGF165),and to transfect pIRES2-EGFP-hVEGF165 into human placental mesenchymal stem cells (HPMSCs) through liposome,and to identify the express activity of hVEGF165 and the multi- differentiation potential of HPMSCs  carrying the target gene. Methods From human leukemia cells HL-60 the gene fragment of hVEGF165 was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR),the pIRES2-EGFP-hVEGF165 recombinant plasmid was constructed,and its correctness was detected by restriction enzyme digestion;the activity of transcription and expression of  HPMSCs  transfected by liposome were respectively detected with RT-PCR,Western blotting and MTT assay; the enhanced green fluorescence protein(EGFP) was observed under fluorescence microscope;and the conditions of transfected target cells of empty vector containing reporter gene EGFP,eukaryotic expression vector carrying reporter gene and target gene were detected;and the multi-differentiation potential of HPMSCs after transfection was indentified.Results The recombinant plasmid pIRES2-EGFP-hVEGF165  was successfully constructed through enzyme cutting identification.The constructed plasmid of HPMSCs after transfection with pIRES2-EGFP-hVEGF165 had transcription and expression activities;the EGFP expression was detected  under fluorescence microscope,and the target gene was successfully transferred into target cells,and the HPMSCs carrying the target gene still maintained multipotentiality.Conclusion The eukaryotic expression plasmid pIRES2-EGFP-hVEGF165 carrying hVEGF165 with expression activity is successfully constructed,and the plasmid is successfully transferred into HPMSCs.The hVEGF165 in HPMSCs has the activities of transcription and expression,and hVEGF165 could promote the proliferation of HPMSCs,and the HPMSCs itself might have the endocrine function of hVEGF165.

Key words: human , vascular endothelial growth factor 165, human placental mesenchymal stem cells, liposome;transfection