Tissue-type Plasminogen Activator Up-regulate Expression Of Vascular Endothelial Growth Factor In ECV304 Cells Via ERK And P38 Signaling Pathway

The construction of a recombinant vector co-expressing the VEGF165 and t-PA genesObjective: To construct a recombinant vector co-expressing the VEGF165 and t-PA genes mediated by pIRES vector, and to lay the basis for the co-expressing gene therapy of deep venous thrombosis.Methods:①Design the primer, then obtain the total RNA from ECV304. cDNA of objective genes were reverse transcripted from total RNA and amplified by PCR.②The full length sequence of VEGF165 gene was amplified and cloned into pMD19-T simple vector to construct pMD19-T-VEGF165KpnI/EcoRI.③Using the method of restriction enzyme digestion step-by-step with PhnI and EcoRI. Digested pMD19-T-VEGFKpn/EcoRI recombinant plasmid and pIRES vector, and recovered purpose fragment, then constructed pVEGF165-IRES.④Purified the PCR amplified products of t-PA gene, and cloned into pMD19-T simple vector to construct pMD19-T-VEGFKpnI/EcoRI.⑤Digested pMD19-T-t-PA XbalI/NotI and pVEGF165-IRES with XbaI and NotI, recovered t-PA gene and pVEGF165-IRES fragment respectivly, then constructed a recombinant vector co-expressing the VEGF165 and t-PA genes.Results:①VEGF165 and t-PA genes were coloned from human umbilical veous endothelial cell line-ECV304 successfully.②A recombinant vector co-expressing the VEGF165 and t-PA genes mediated by pIRES vector were constucted successfully.③After transfected vascular endothelial cells in vitro, we got little protein expression of t-PA gene than VEGF165, the rate is 30.6%, not reach the demands of this experiment.Conclusios: The protein expression of downstream gene in a recombinant vector co-expressing two genes mediated by pIRES vector is very low, the expressio rate of this recombinant plasmid is not equal after transcription. It's necessary to investigate how to increase the expression of dowmstream gene and prolong the duration of expression of this co-expression recombinant vector, then increase the therapeutic effect.Construction of human tissue-type plasminogen activator on its adenovirus expression vectorsObjective: To construct a human tissue-type plasminogen activator on its adenovirus expression vectors.Methods: t-PA gene was coled to adenoviral shuttle plasmid-pAdtrack-CMV to contruct pAdtrack-CMV-t-PA. After linerized pAdtrack-CMV-t-PA, we transform it into E.coli BJ5183 competence bacterium which contained adenoviral skeleton plasmid (pAdeasy-1) to make homologous recombination in cell. The recombinant adenoviral vector carrying t-PA gene was transfected into HEK293 cells to package the adenovirus. Then amplified the recombinant adenoviral vector, observed the GFP expression under fluorescence microscope and followed by PCR and restriction enzyme digestion identification.Results: With the methods of PCR and restriction enzyme digestion identification and GFP expression, it was confirmed that the Adt-PA was constructed successfully. After amplification, the virus titer was reached to 2.56×109pfu/ml thus reached the demands of research remainedConclusions: The Adt-PA was constructed successfully and the virus titer was reached the demands of research remained after amplification and purification. Expression of VEGF165 in ECV304 cells induced by tissue-type plasminogen activatorObjective: To investigate the expression of VEGF mRNA and protein in ECV304 cells transfected by Adt-PA in vitro and identify whether extrinsic source t-PA can increase the expression of VEGF.Methods: Transfected active ECV304 cells with different virus titers, trasfection ratio were 1:10, 1:50 and 1:100 expectively, observed the cells proliferation and selected the fitting MOI value. Then transfectd ECV304 with Adt-PA and investigated the expression of the t-PA protein with Westernblot analysis, divided ECV304 cell to two groups, one was transfected by Adt-PA and the other only by Ad used as control. After mixed culture for 24 hours, RT-PCR was used to evaluate the VEGF mRNA expression and Westernbolt analysis was used to detect the VEGF protein expression.Results: Adt-PA had some promotion effects on proliferation of ECV304 at 1:50 , but the proliferation decreased at 1:100. After transfection, the quantity of t-PA expression increased gradually within 72 hours. The expression of VEGF mRNA and protein increased obviously at 24h and 48h compared with control group, this difference have significance (p<0.05).Conclusions: Extrinsic source t-PA can promote VEGF mRNA transcription and protein expression in ECV304 cells.t-PA Up-regulates VEGF Expression in ECV304 Cells Via ERK/P38 Signaling PathwayObjective: To investigate the role of the ERK/P38 signal transdution pathway in the effect exerted by extrinsic t-PA on VEGF expression in ECV304.Methods: Divided ECV304 cells into two groups, one is transfected by Ad and the other is transfected by Adt-PA, Westernblot analysis phosphorylation of ERK/P38/JNK in these two groups at 0h, 24h, 48h and 72h. Inorder to identify the effects on t-PA up-regulate the expression of VEGF in ECV304, we used PD98059, a special blocking agent of ERK signaling pathway. Grouping like below: Ad, PD, Adt-PA and Adt-PA+PD. Westernblot analysis the expression of VEGF and phosphorylation of ERK. Also in order to identify the effects on t-PA up-regulate the expression of VEGF, we used SB203580, a special blocking agent of P38 signaling pathway. Grouping and Westernblot analysis were like above. At last, we used PD and SB affected ECV304 cells, Westernblot analysis the phosphorylation of ERK/P38 to identify the cascade connection of these two signaling molecule.Results: After transfected by Adt-PA, ERK and P38 could be activated obviously in ECV304. This activation manifested a time dependent mammer excepted JNK. The difference of pERK and p-p38 between 24h, 48h and 72h in these two group have significance. While PD and SB can decreased the expression of VEGF in ECV304 cells, the same were pERK and p-p38 (p<0.01). Phosphorylation of ERK induced by Adt-PA transfection could be suppressed by PD but not SB, while phosphorylation of P38 induced by Adt-PA transfection could be suppressed by SB also PD.Conclusions: ERK and P38 were cascade signaling pathway during the up-regulation of VEGF expression after ECV304 transfected by Adt-PA. ERK at the upstream of this pathway, and this cascade signaling conduction were identified to play a very importent role.