Experimental Study Of The Effects Of Bone Marrow Mesenchymal Stem Cells Transfected With Hypoxia-inducible Factor-1α Gene For The Ischemic Heart Disease

Ischemic heart disease(IHD)is the disease that the myocardial blood-deficiency occurs when the coronary artery blood circulation can not match the heart's need.IHD seriously damages mankind's health due to its high morbidity and mortality.The basic pathology is characterized by the massive cardiaomyocytes irreversible loss,which is replaced by non-contractile fibrous cells.Ventricular remodeling process develops in the remaining surviving myocardium if the ischemia persists,and eventually the progressive deterioration of congestive heart failure is inevitable.Current therapy including drug treatment,percutaneous coronary intervention(PCI)and coronary artery bypass graft(CABG) mainly aim at saving the viable cardiomyocytes,improving the function of survival cardiomyocytes and reversing ventricular remodeling.The ability to regenerate damaged myocardium presents a major challenge in the treatment of myocardium infarction.Therapeutic angiogenesis,which have become leading fields in the research of IHD therapy worldwide,is a potentially viable therapeutic strategy designed to amplify the native angiogenic process and enhance the reperfusion for reversing the ischemic remodeling process,compensate for the loss of ventricular mass and contractility,eventually restore cardiac function.There are 3 approaches to induce angiogenesis and vasculogenesis:recombinant protein,cells and gene therapy.Hypoxia-inducible factor-1α(HIF-1α)is a protein with transcription activity.HIF-1 can not only promote the transcription and expression of many angiogenisis factor,such as VEGF and TGF-β,to increase the regeneration of vesscle and enhance the reperfusion,but also improve the adaption of ischemic myocardial cell in anoxia condition,intensify the process of zymolysis,and supply myocardial cell with more energy.So it is possible that the therapeutic effect of HIF-1 would be better than that of VEGF or FGF treatment simply.Bone marrow mesenchymal stem cells(MSCs),the early cell of mesoderm,which can keep the potent plasticity of differentiate to multi-tissue have the advantage of can be isolated easily from a variety of sources,genetic stability,no immunologic and ethical concerns.So they were considered as ideal candidate donor cells for stem cell therapy and target cells for gene transfer.Thus we put forward the hypothesis that the combination of MSCS transplantation and Hypoxia-inducible factor-1α(HIF-1α)gene transfer might improve the function of gene or cellular treatment mutually and would be superior to either strategy alone for treatment of chronic myocardial ischemia.In this study,we investigate the effects of MSCs transferred with HIF-1αgene transplantation for IHD therapy in rat myocardial ischemia model and provide experimental principles for further clinical application for human ischemic heart disease.This experimental study is composed of three parts.PartⅠGene cloning of HIF-1αand construction of the HIF-1α-pcDNA3.1 expression plasmid.Objective:To construct and identify eukaryotic expression vector of human HIF-1α(HIF-1α-pcDNA3.1).Method:HIF-1α- pcDNA3.1 is constructed with the technology of gene recombination.Then the recombined plasmid is identified by enzyme digesting and DNA sequencing.Result:HIF-1α-pcDNA3.1 eukaryotic expression vector is constructed successfully.PartⅡExpression of HIF-1αgene in Rat mesenchymal stem cells in vitroObjective:To investigate transfection and HIF-1αgene expression of HIF-1α-pcDNA3.1 eukaryotic expression vector in cultured mesenchymal stem cells.Methods:MSCs of Wistar rat was isolated by density gradient centrifugation and purified on the basis of their ability to adhere to plastic. Detections of cell surface marker phenotype,including CD34,CD45, CD44 and SH3(CD73),were performed using flow cytometry.The vector HIF-1α-pcDNA3.1 was transfected into MSCs with the method of lipofectamine mediated.The expression of HIF-1αin the transfected cells was detected by RT-PCR,ELISA and Western blot analysis.Results:1.MSCs were CD34~-,CD45~-,CD44~+,SH3~+.They can differentiate into bone and adipocytes successfully.2.The expression of HIF-1αgene in the transfected rat MSCs was demonstrated by RT-PCR, ELISA and Western blot analysis. PartⅢEffects of myocardial transplantation of MSCs transfected with HIF-1αgene for heart functionObjective:To evaluate the effect of MSCs transfected with HIF-1αgene for heart function restoration and mitigation of the adverse effects of ventricular remodeling after myocardial infarction,compare the therapeutic difference between the combinated therapy and single cell or gene therapy.Methods:Wistar rat ischemic heart animal model was constructed by left anterior descending coronary artery ligation.80 ligated animals were divided into 5 groups(16 each).2 weeks later,4 groups were injected at the heart infarct zone with HIF-1α-transfected MSCs(Combo group),MSCs(Cell group),HIF-1α-pcDNA3.1(Gene group),or medium (Control group).The left one(Model-assessment group)was served as model assessing.Another 16 rats without ligated were used as a sham operation group.4 weeks after the injection,the rat cardiac function was measured by Buxco system.Brdu labeling andⅧfactor were identified by immunohistochemical stain;size of infarcted area was calculated by Evan's blue stain.HIF-1αgene expression was evaluated by RT-PCR.Results:78.4%(80/102)of the ischemic models,constructed by coronary artery ligation,were qualified for the next study.2 weeks after the ligation,the animals had smaller infarcted area(P＜0.05)and better heart performance than those of 6 weeks'.4 weeks after the transplantation,the combo group animals' infarcted heart size was (29.8±3.2)%,smaller than that of cell and gene group's(P=0.049,0.016), which were smaller again when compared with control group;the heart function,measured by Buxco system,was better than that of both cell and gene groups',the capillary density was similary to gene group's but more than cell group.Brdu and Troponin-T double stain showed that there was a different increase of survived cardiaomyocytes,some were double stain positive,located at the infarcted area of combo,cell and gene group.The expression of HIF-1αin combo group was higher than that of other groups.Conclusions:1.HIF-1αwas constructed into the pcDNA3.1 vector successfully. There are no mutations by sequencing confirmation.2.HIF-1αhad been successfully transfected into MSCs and well expressed demonstrated by RT-PCR,ELISA and Western blot analysis..3.Transplantation of MSCs transfected with HIF-1αgene can improve myocardial perfusion and restore heart function.The effects was better than that of cellular or gene therapy simply.