| Background:Vascular endothelial cells play a key role in the regulation of vascular function and homeostasis.The vascular endothelial dysfunction is identified as one of major causes of vascular injured disease.The main treatment direction for vascular disease is to restore vascular endothelial function.Cell and gene therapy have been proved to be effective to promote neovascularization in various animal models.Gene transfected to the different components of the vessel wall can improve endothelial function and become a potential therapeutic strategy for cardiovascular diseases.Because of the defect of expression vector and limited restoration endothelial cells function,gene therapy can not completely restore endothelial cell function. In recent years,stem cells transplantation have become a new protocol in the treatment of a variety of diseases,which can replace,or repair damaged tissues or organs to strengthen their biological function.Stem cells can maintain the undifferentiated status for the proliferation and differentiate into various cell types in vitro.A number of studies have found endothelial progenitor cells(EPCs),settled in bone marrow and peripheral blood,EPCs can express characteristic antigen of endothelial cells and differentiate into endothelial cells in vitro.Also,EPCs can differentiate into endothelial cells in vivo micro-environment and restore some of the endothelium functions.Burns or vascular impaired induced cytokines release such as vascular endothelial growth factor (VEGF),fibroblast growth factor(FGF).EPCs stimulated by cytokines in the bone marrow can move into peripheral blood and participate vascular endothelial regeneration.But differentiation need local micro-environment.One of particularly important cytokines is VEGF.Therefore,given these new concepts and experimental studies,combination of gene and stem cell therapy may enhance recovery of injured endothelium.VEGF gene transfected to the EPCs in vitro enhanced its homing to the intima of impaired vessel and differention to endothelial cell and further enhanced EPCs therapeutic effects on vascular intima impaired disease. EPCs can be easily obtained from the bone marrow compared to the embryonic stem cells and skeletal muscle stem cells.EPCs can differentiated into endothelial cell.This study isolated and cultured rat's bone marrow-derived EPCs in vitro.PcDNA3-hVEGF165 plasmid was constructed and transfected to EPCs.The effect of gene modification on EPCs function including the migration,adhesion,proliferation was observed.Gene-modified EPCs were transplanted into artery intima injury model.It was observed that gene-modified EPCs migrated to the intima of impaired vessel and differentiation to endothelial cells.The function of gene transfer EPCs was evaluated by vascular injury partial re-endothelial model.The gene and stem cell therapy were combined in treating injured vascular intima rehabilitation.Discussion of EPCs by VEGF gene transfection on vascular biological function,try to provide some theoretical basis for the vascular endothelium restoration.Methods:1.Constructed the eukaryotic expression vector with hVEGF165 gene:hVEGF165 gene was extracted from plasmid pGEM-hVEGF165 and inserted into pcDNA3 vector. Constructed eukaryotic expression vector contains hVEGF165 gene.Recombinant plasmid were verified by enzyme digestion and sequence analysis.2.EPCs culture,purification,characterization:separate mononuclear cell from rat bone marrow by density gradient centrifugation method.The ultrastructure of EPCs was observed by Transmission electron microscopy.Cells expressed double FITC-UEA-I and Dil-acLDL fluorescence were identitied as EPCs.3.VEGF gene was transfected to EPCs,gene-modified EPCs adhesion,migration function was evaluated.VEGF level was confirmed by ELISA method and cell proliferation activity by MTT method.4.After the artery intimal vascular injury model was established,gene-modified EPCs was transplanted into experimental rats.It was evaluated that EPCs migrated to intima of impaired vascular,with VEGF gene expression and its differentiated into endothelial cells, influence of impaired vascular re-endothelial by gene-modified EPCs.Results:1.The mammalial expression vector,pcDNA3-hVEGF165,were verified correctly by enzyme digestion and sequence analysis.PcDNA3-hVEGF165 can be used to transfected eukaryotic cells. 2.EPCs purification,culture,identification:mononuclear cells separated from rat bone marrow by density gradient centrifugation method.Adherent cells gradually increased,with a paving stones shape in cultured 7 days.With DiI-acLDL and FITC-UEA-I staining, fluorescence microscopy revealed yellow cells expressed double DiI-acLDL and FITC-UEA-I fluorescence,vWF immunohistochemical staining in brown-positive cells confirmed the differentiating EPCs.3.Detection of VEGF expression of gene-modified EPCs by ELISA method.VEGF expression of EPCs transduced with pcDNA3-hVEGF165 was statistically significant comparison with nontransduced EPCs(p＜0.01).Gene-modified EPCs improve its adhesion and migration.Proliferation activity of EPCs transduced with pcDNA3-hVEGF165 was statistically significant compared with nontransduced EPCs(p＜0.05).4.Rat femoral artery intimal vascular injury model was established by flexible guide wire.Gene-modified EPCs was transplanted into rats and DiI-acLDL staining showed that gene-modified EPCs can adhere,survive in intimal vascular injury site and promote re-endothelial.Conclusion:1.Construction of the eukaryotic expression vector pcDNA3-hVEGF165 containing human VEGF165 gene sequences can be used in VEGF165 gene eukaryotic expression studies.2.High expression of VEGF was observed in Gene-modified EPCs.3.VEGF gene-modified EPCs improve its adhesion,migration and proliferation.4.Gene-modified EPCs can migrate to impaired intima and survive in that sites in vivo. Gene-transfected EPCs have stronger intimal repairing ability compared with non-transfected EPCs.The same number of transplantated,gene-modified EPCs have also stronger ability to restorate normal vascular endothelial structure. |
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