Experimental Study Of Human VEGF165 Gene Transfection Into Endothelial Progenitor Cells From Human Peripheral Blood 作者姓名 赵亲明

Objective With the population aging and changes in diet, Lower limb ischemic disease has become a common and frequently-occurring disease to older persons.It has been the main reason for amputation disability.At the same time,it has become a difficult and hot research spot of vascular surgery.The research of promoting organization angiogenesis has developed as a new method to treat the ischemic disease. There are three approaches to induce angiogenesis and vasculogenesis:proteins,cells and gene therapy. Endothelial progenitor cells and vascular endothelial growth factor are play an important role in angiogenesis and vasculogenesis. This study intends to combine the gene therapy with the stem cell therapy, To investigate the feasibility of human VEGF165 gene transfection into endothelial progenitor cells (EPCs) derived from human peripheral blood, and the influence of proliferation and the ability of secreting VEGF and NO on EPCs.Methods The mononuclear cells (MNCs) were collected from the human peripheral blood, which is mobilized after rhG-CSF, isolated by the density gradient centrifugation and cultured in the fibronectin-coated culture plates with M199 medium. At the 7th day, the adherent cells were identified by two methods:(1) The adherent cells's morphology was observed under the inverted microscope.(2) The cells were observed by the laser scanning confocal microscope (LSCM) after double stained with FITC-UEA-I and DiI-acLDL.Then the pcDNA3.1-hVEGF165 plasmid was amplificated and extracted,and identified by double-digestion method and sequencing. The experiment divided into three groups:EPCs transfected with pcDNA3.1-hVEGF 165 by liposomal, EPCs transfected with pcDNA3.1 by liposomal and EPCs without transfection. After transfection the expression of VEGF and NO in culture supernatants were each detected by using ELISA and nitrate reductase method.The proliferation of EPCs after gene transfection was detected by MTT methods.Results Cells expressed double FITC-UEA-I and Dil-ac-LDL fluorescence were EPCs.The expression of VEGF and NO in the medium of EPCs transfected with pcDNA 3.1-hVEGF165 were obviously more than the other two groups(P<0.01). No influence of EPCs proliferation could be found after transfection.Conclusion EPCs from human peripheral blood could be successfully transfected with VEGF 165 gene,and expressed a certain concentration VEGF protein,and could promote the secretion of NO, and no influence of EPCs proliferation could be found after transfectioni. It was provided experimental evidence to further study of the combination treatment of VEGF 165 gene and EPCs to ischemic disease.