Studies Of Transplantation Of Endothelial Progenitor Cells Transfected By VEGF Gene Into Ischemic Myocardium

Background: Ischemic heart disease (IHD) is a major disease that seriously threatens human health.IHD is characterized by inadequate blood flow and tissue oxygen supply that is typically theconsquence of decreased patency of atherosclerotic vessels. The infarctic zone graduallyreplaced by scar tissue which have much influence on the systolic and diastolic function ofthe heart. The myocardial remodeling often cause congestive heart failure. Traditionally,patients with IHD requiring a revascularizing procedure have had undergo either medicinetreatment or percutaneous transluminal ballon angioplasty, or coronary artery bypass surgery.Despite the fact that advances in modern medical, surgical and interventional treatment ofthe disease have produced significantly improved results, the efficacy of all these methods islimited in patients with diffuse distal coronary artery disease. The method of alleviatemyocardial remodeling by implanted cells and transfected special angiogenic growth factorgenes to enhance the angiogenesis may present a new approach to the treatment of ischemicheart disease. Up to now, VEGF and FGF have been the most studied factors in the field oftherapeutic angiogenesis. In our study, we evaluated the effect of EPCs transfected byAd.VEGF165 transplantated into ischemic myocardium on angiogenesis and myocardialfunction, as an alternative to angiogenic gene or protein therapy. Firstly, we constructed areplication-deficient adenovirus vector coding for the gene of VEGF165, and induce rat bonemarrow endothelial progenitor cells. Then we transfected EPCs with Ad.VEGF165 in vitroand tested the efficiency of transfection and protein expression. The proliferation anddifferentiation effect of Ad.VEGF165 on EPCs were certified. Finally, we transplanted theEPCs transfected by Ad.VEGF165 into rat ischemic myocardium and find the survival of thetransplanted cells and the ability to increase the growth of neovascularization in ischemiczone which enhance local blood perfusion and subsequently improve myocardial function. - 5 -第二军医大学博士学位论文 英文摘要 胸心外科专业Methods:1. The construction of replication-deficient recombinant adenovirus coding for VEGF165gene (Ad.VEGF165) Human embroynic kidney 293 cells were transfected with DNA-TPC and recombinantadenovirus cosmid vector pAxcAwt.VEGF165 by the calcium phosphate method. Virus cloneswere isolated and propagated for restriction analysis. The desired Ad vectors were purifiedby density gradient ultracentrifuge and titrated in 293 cells.2. The inducing and culture of rat bone marrow endothelial progenitor cells Bone marrow cell suspension of adult rat was prepared by discontinuous gradientcentrifugation on Ficoll, mononeuclear cells in the middle layer was collected, cultured for24 hours, then suspending cells were put into selective culture medium and cultured in thefibernectin coated plate. The cells was observed under inverted microscope every day. Thecells cultured were identified by transmission electron microscope, flow cytometer,immunohistochemical staining with anti-factor Ⅷ, CD31, CD34, VRGFR-2 antigen, and theability of secreting nitrous oxide and combining BS1-Lectin.3. Transfection of Ad.VEGF165 to EPCs and its effects of proliferation and differentiationon EPCs in vitro. EPCs were induced and cultured. Transfection efficiency of adenovirus vector toEPCs were identified by infection with various titrations of Ad.LacZ. After Ad.VEGF165transfection, VEGF protein expression in EPCs and its secretion in culture medium weremeasured by methods of immunohistochemical staining, Western-blot and ELISArespectively. The proliferation effect of Ad.VEGF165 on EPCs were measured by MTT assay.Finally, we also examined the differentiation effect of Ad.VEGF165 on EPCs.4. The research of VEGF-expression EPCs transplanted into ischemic myocardiu...