The Influence On Proliferation Of Which Human Umbilical Vein Endothelial Cell Co-transfected By UPA And VEGF165Single Eukaryotic Plasmids

Objective:To construct urokinase-type plasminogen activator (uPA) and human vascularendothelial growth factor165(VEGF165) single gene eukaryotic expression plasmid,and then they were co-transfected into human umbilical vein endothelial cells(HUVECs), to detect uPA, VEGF165expression and proliferation of HUVECs aftertransfection.Methods:1.Construction of uPA gene and VEGF165single gene eukaryotic plasmid:Design and synthesize primers with restriction sites, introduce new restriction sites toVEGF and uPA gene, which were directed to connect into the eukaryotic expressionvector pIRES2-EGFP, and then transformed in-to competent cells. Identifyrecombinant plasmids pIRES2/EGFP-VEGF165and pIRES2/EGFP-uPA by PCR，restriction analysis and DNA sequence,.2. Transfect human umbilical vein endothelial cells by liposome after ampl-ification of recombinant plasmid, and divide into following five groups in vitr-o：A group (PBS without stimulus),B group (transfected with the empty plasm-id pIRES2/EGFP), C group (pIRES2/EGFP-uPA+pIRES2/EGFP co-transfected),D group (p-IRES2/EGFP-VEGF165+pIRES2/EGFP co-transfected), E group(pIRES2/EGFP-VEGF165+pIRES2/EGFP-uPA co-transfection).Detect proliferation in each group by MTT after transfection, and describe the growth curve; U-sing real-time quantitative PCR (QPCR) to detect expression of mRNA VEGF165and mRNA uPA; Western blot to detect the expression of VEGF165anduPA.Results:1.Identification of pIRES2/EGFP-VEGF165and pIRES2/EGFP-uPA: Obtained VEGF165and uPA bands by agarose gel electrophoresis after digestion ofrecombinant plasmids by XhoI and SalI，We found digested bands and polymerasechain reaction products in the same position electrophoresis,meanwhile we confirmedthat the gene insert in the right direction and no sequence mutation by PCR,restriction analysis and DNA sequencing.2. There was significant difference (P<0.05) that MTT showed proliferation ratein group E was higher than A, C, B groups, but between groups D, E there were nosignificant difference (P>0.05). QPCR showed relative of VEGF165mRNA in groupE was higher than in A, B,C groups, and there was a significant difference (P <0.05),meanwhile between group D and E there were no significant difference (P>0.05). Onthe other hand relative of uPA mRNA in group E was higher than in A, B, D groups,there was a significant difference (P <0.05),and between groups C and E there wereno significant difference (P>0.05).What Western blot showed was that VEGF165inD, E groups were higher than A, C, B group. And uPAin C, E group were higher thanA, B, D groups, and between groups C and E there were no significant difference,uPAin group D was higher than A, B groups.Conclusion:VEGF165and uPA single eukaryotic expression plasmids are cotransfectedwith an effective expression in human umbilical vein endothelial cells,More over wefind proliferation rate increased significantly and uPA is promoted to express in cellsupregulated VEGF165.We achieved a nice theoretical basis.for treatment of venousthromboembolism.