Experimental Study of the Effects of Bone Marrow Mesenchymal Stem Cells Transfected with Sonic Hedgehg Gene for the Chronic Ischemic Heart Disease
|School||Central South University|
|Keywords||mesenchymal stem cell shh gene transfect ischemic heart disease cell therapy gene therapy regeneration angiogenesis|
Coranary heart disease, the main cause of congestive heart failure presents an increasing morbidity rate in China. After the myocardial infarction, lots of cardiaomyocytes irreversible lost, which eventually replaced by fibrous cells. Although hypertrophy and cell division occurs in the border area of the dead tissue, it is not sufficient to stop the progress of congestive heart failure. The use of gene and cellular therapy offers a promising approach to prevent and cure the heart failure.Bone marrow mesenchymal stem cell (BMMSC), that persents in the adult bone marrow tissue is the early cell of mesoderm, have the ability to self-renew and differentiate into kinds of tissue cells and the advantage of being able to be easily isolated from a lot of sources, no immunologic, genetic stability and ethical concerns. So they were considered as excellent candidate donor cells for target cells for gene transfer and stem cell therapy. We put forward the hypothesis that the combination of shh gene transfer and BMMSC transplantation would be superior to either strategy alone for the treatment of chronic myocardial ischemia.This experimental study contains two parts. Part I is the study of the expression of SHH gene in rat BMMSC in vitro. We isolated and purfied BMMSC with density gradient centrifugation-adherent method. Then, these cells were transfected with pcDNA3.1-shh by electrotransfection. Part II is the study of the effect of transplantation of shh-expression BMMSC for heart function. Wistar rats’left anterior descending coronary artery were ligated,4 weeks later, they were injected separatly with shh gene modified rat BMMSC, unmodified BMMSC, shh gene, shh gene and unmodified BMMSC or DMEM medium at the heart infarcted zone. The treat effect was evaluated by heart function and electric mirror.PartⅠExpression of shh Gene in Rat mesenchymal stem cells in vitroObjective:To evaluate the feasibility of shh gene transfection into mesenchymal stem cells.Methods:1. BMMSC 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 antigens, including CD34, CD45, CD44 and SH3, were performed using flow cytometry.2. The pcDNA3.1-shh was transfected into BMMSC with the method of electrotransfection. The expression of shh in the transfected cells was detected by Western blot analysis.Results:1. BMMSCs were CD34-, CD45-, CD44+ and SH+.2. The expression of shh in the transfected rat BMMSC was demonstrated by Western blot analysis.Conclusions:The experiment demonstrated that shh had successfully expressed in BMMSC. Part II The Effect of Bone Marrow Mesenchymal Stem Cells Transfected With shh Gene for Heart Function After Myocardial Infarct on RatObjective:To evaluate the effect of BMMSC transfected with shh gene for heart function restoration 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. The ligated animals were divided into 5 groups (10 each).4 weeks later,4 groups were injected at the heart infarct zone with shh-transfected BMMSCs (A group), BMMSCs (B group), shh gene (C group), shh gene+BMMSCs(D group)or medium (E group). 1,2,4and8 weeks after the injection, the treat effect was evaluated by heart function.Results:After the left anterior descending coronary artery ligation, the heart function of the E group has been decreasing continuously,while the other 4 groups do not change significantly,and also shows no significant different among A,B,C,D groups.Conclusions:Transplantation of BMMSC transfected with shh gene can prevent the heart function decreasing continuously,but not better than the combinated therapy and single cell or gene therapy.