Transplantation Of Endothelial Progenitor Cells Transfected With VEGF165 Gene In The Treatment Of Chronic Deep Venous Thrombosis

Objective: To study the co-therapeutic effect of stem cell transplantation and gene transfection on the chronic deep venous thrombosis.Methods: The recombinant adenoviral vector carrying the VEGF165 gene was constructed by pAdEasy system.pAdEasy-VEGF165 transfected into HEK293A cells to package the adenovirus,followed by identification by means of GFP.PCR and gene sequencing identify the recombinant.EPCs were isolated from rat bone marrow by Ficoll and cultured with EBM-2MV medium cultivation.MTT assay were used to draw growth curve of cells.After infection with the recombinant Ad-VEGF165,EPCs were examined with a fluorescent microscopy and ELISA for their expression of VEGF,MTT assay was employed to assess the growth character,and Laser scanning confocal microscope for transduction efficiency.Experimental rat model of chronic deep vein thrombosis were obtained by partial ligation of inferior vena cava.The rats were randomly divided into four groups:A(n=25),Ad-VEGF165 transfection group,1ml 106 Ad-VEGF165-EPCs transplantation;B(n=25),EPCs group,1ml 106 EPCs transplantation;C(n=25),Ad-GFP transfection Group, 1ml 106 Ad-GFP-EPCs transplantation; D(n=25),control group, 1 ml of medium transplantation.After transplantation, real-time quantitative PCR was used to detect VEGF, ANG-1 mRNA expression level and western blot of inferior vena cava thrombosis, and VEGF,ANG-1 protein expression changes.HE staining and Immunohistochemical staining was performed to detect recanalization and the neovascularization was observed by scanning electron microscopy.The capillary density was determined quantitatively by counting the capillaries under high-power microscope.SPSS11.5 software used for analysis, P <0.05 for the difference was significant.Results: Ad-VEGF165 was constructed and successfully cultivated a bone marrow-derived endothelial progenitor cell(sEPCs).Appropriate ratio of transfection had promotion of the growth of EPCs.After transfection, EPCs had secretioned VEGF protein.EPCs in vivo survival,differentiated into endothelial cells was viewed by marker of Dil stain after transplantation. VEGF, ANG-1 mRNA were expressed in A, B, C and D group after transplantation, and that were significantly increased in group A than group B, C and D (P<0.05), were significantly increased in group B and group C than group D (P<0.05), were no statistical significance in between group B and group C. The recanalization capillary density were significantly higher in group A than that in group B,C(P<0.05),than in group D(P<0.01) ,were significantly higher in group B and group C than in group D(P<0.05).And there was no statistical significance in between group B and group C.The neovascularization were identified by immunohistochemical staining of vWF antibody, that were compose of endothelial cells.Conclusion: EPCs were transfected by Ad-VEGF165 successfully.A suitable ratio of transfection can improve the efficiency of EPCs.That can improve potentiality of promotion of angiogenesis after transplantation. And had accelerate organization and recanalization of vein thrombus.