Study Of Construction Of PcDNA3.0-hVEGF165 And Transfection On Endothelial Progenitor Cell Derived From Bone Marrow In Vitro

Objective :To construct a human vascular endothelial growth factor (165) eukaryotic expression plasmid: pcDNA3.0-hVEGF165 and to investigate the study of transfection on endothelial progenitor cell derived from rat,s bone marrow in vitro.Methods:Design the primer, then obtain the total RNA from ECV304. hVEGF,s cDNA was reverse transcripted from total RNA and amplified by PCR, then it was inserted into the vector pcDNA3.0. The recombinant plasmid of pcDNA3.0-hVEGF165 was identified by PCR, restriction enzyme digesting and sequencing. We obtain the rats, EPC by culturing the mononuclear cell isolated from bone marrow in EGM-2MV, and the cell we acquired could express those distinctive surface markers, such as CD34, CD133, FLK-1. pcDNA3.0-hVEGF165 transfect EPCs mediated by liposome .Detect the VEGF protein level in the cultural medium supernatant after VEGF transfection by ELISA. Evaluate the effect of secreted VEGF by stimulating ECV304 growing. Evaluate the influence of EPCs, activity after transfection through MTT. Detect the expressive changes of EPC by immunocytochemistry , such as VEGF, vWF, VEGFR-2(FLK-1).Results: The recombinant eukaryotic expression plasmid pcDNA3.0-hVEGF 165 was successfully constructed. It is a well method to enrich EPCS by culturing mononuclear cell in EGM-2MV, and the EPCS obtained could express those distinctive cell surface markers such as CD34, CD133, FLK-1 and so on. EPCS could excrete a certain concentration of VEGF protein after transfection mediated by liposome, and the concentration of every 24 hours reached the peak in the forth day, then began degrading. No influence of EPC,S proliferation was found after transfection. VEGF gene transfection might be helpful for EPC to maintain the characters of endothelial cell. The rate of EPC after VEGF gene transfection expressing the markers of VEGF,vWF,VEGFR-2(FLK-1) became higher and higher within 7 days after transfection.Conclusions: We successfully construct the eukaryotic expression plasmid pcDNA3.0-hVEGF165. The EPCS transfected by pcDNA3.0-hVEGF165 mediated by liposome could excrete functional VEGF protein. The EPCs proliferation was not influenced by transfection. It is helpful for the EPCs to maintain the characters of endothelial cell after VEGF transfection.