Experimental Study Of The Effects Of Bone Marrow Mesenchymal Stem Cells Transfected With Sonic Hedgehog Gene For The Acute Ischemic Heart Disease

Acute myocardial infarction (AMI) is one of the major cause of heart failure and death in the country and its incidence is also rising with the development of our living standard.After the acute myocardial infarction, the significant loss of the Myocardial cells is inevitable,and this led to low cardiac function after myocardial infarction.In spite of a small number of the stem cells out of the heart and myocardial cells remained can growth and differentiation,however,this limited repair is not sufficient to alleviate the heart failure after acute myocardial infarction.At present, the best treatment for Heart failure is heart transplantation,but as the reason of the scarce source, long time of immune suppression treatment and the high cost of heart transplantation,let the heart transplantation as a conventional treatment programs impossible.Against this background,the stem cell therapy and gene therapy ischemic heart disease has gradually become a clinical research hot spots.Bone Marrow Mesenchymal Stem Cell(BMMSC) is the early cell exist in adult bone marrow tissue,it can keep "self-renewing" and has feature of differentiate to multi tissue with the advantage of can be isolated easily from a rich sources, genetic stability, no immunologic and ethical problems.Such advantages qualified BMMSC donor cells for stem cell therapy and gene therapy. In the meantime, After transfected with exogenic gene by different vectors, these cells can keep their differentiate capacity and genetic stability resulting in long-term and stable gene expression and better performance of incoporation into surrounding tissue. We put forward the hypothesis that the combination of BMMSC transplantation and Sonic Hedgehog(Shh) gene transfer would be superior to either strategy alone for acute myocardial ischemia treatment.The experiment was divided into two sections.SectionⅠ,we used Sigma-Aldrich 10771 to isolated and purfied Wistar rat BMMSC,then the cells were transfected Sonic hedgehog gene with Nucleofector.And after that we will test the gene expression.SectionⅡ,we studied the effect of transplantation of Shh-expression BMMSC for heart function. We studied heart functions and other indicators of the rats after the cells injection for 1 week,2weeks,4weeks,8weeks and compare with cell therapy and/or gene therapy. Objective:To evaluate the possibility of Sonic hedgehog gene transfection into BMMSC.Methods:1. BMMSC of Wistar rat was isolated by Sigma-aldrich 10771 and purified on the function of their ability to adhere the inner-wall of the flask. Detections of cell surface antigens:CD34, CD44, CD45 and SH3.2.The pcDNA3.1-Shh was transfected into BMMSC with the method of electroporation apparatus-Nucleofector. The expression of Sonic hedgehog gene in the transfected BMMSC cells was detected by Western blot analysis and other Detection.Results:1. BMMSCs were CD34, CD45, CD44+ and SH+.2. The expression of Shh gene in the transfected BMMSC was demonstrated by Western blot analysis and Electron microscope detection.Conclusions:The experiment demonstrated that Sonic hedgehog gene had successfully expressed in BMMSC. Objective:To evaluate the effect of BMMSC transfected with Shh gene for heart function restoration and mitigation of the adverse effects of ventricular remodeling after Acute Ischemic Heart Disease,then compare the therapeutic difference between the combinated therapy and single cell and/or gene therapy.Methods:200 acute ischemic heart animal models were made by left anterior descending coronary artery ligation. The models were divided into 5 groups.Then we immediately inject with Shh-transfected BMMSCs (T), BMMSCs (B), pcDNA3.1-Shh DNA (G), mixture of BMMSCs and pcDNA3.1-Shh DNA(M), DMEM (C) at the heart infarct zone.1,2,4,8 weeks after the injection, the rat cardiac function was measured by Buxco system(buxco XA). Electron microscope and Immunohistochemistry test were used to detect the ultrastructure change.Results:About 80% of the acute ischemic models, constructed by coronary artery ligation, were qualified for the following step.1 and 2 weeks after the transplantation, the Transplantation group animals' LVSP and +dp/dt have the trend to be better than the Control group. The Transplantation group animals' LVSP is better than the Control group in4,8weeks group.The electron microscope observation and Immunohistochemistry shows BMMSC survival and change to myocardial cells.Conclusions:Transplantation of BMMSC transfected with Shh gene can help the restoration of heart function.