Experiment Study On Treatment Of Heart Infarction Model Rat By Activation Notch Signal Of Bone Marrow Mesenchymal Stem Cells Implanted Into Infarction Myocardium

Background Viablec ardiomyocytes after myocardial infarction(MI) are unable to repair the necrotic myocardium due to their limited capability of regeneration.Coronary artery occlusion caused myocardial infarction,followed by cardiomyocytes death,fibroplasia,scar formation and cardiac remodeling due to ischemia.Heart function abruptly decreased.Transplantation of bone marrow mesenchymal stem cells (BMMSCs) increased the number of myogenic cells in myocardial infarction tissue and improved heart function.Level of serum vascular endothelial growth factor(VEGF) increased at acute phase of myocardial infarction.It may enhance angiogenesis around myocardial infarction tissue.Notch signal is one of the main direct action signal path between cell and cell,and is key point to determine cell fate of organisma multicellularis.The Intracellular Domain of Notch(ICN) is activity style of Notch signal,Notch signal is activated without ligand through transfection of ICN by carrying agent mediated.It provide convenience to study and research for biological function of Notch signal.Purpose we designed the experiment to probe the efects of capillary density and heart function through BMMSCs transfected ICN gene on cardiac remodeling after myocardial infarction.At present,The influence of BMMSCs transfected ICN gene on cardiac remodeling after myocardial infarction on the heart reconstitution is not very clear.we designed the experiment to probe the influence of the heart reconstitution through BMMSCs transfected ICN gene on cardiac remodeling after myocardial infarction. ntroversial.In order to investigate whether the implantation of BMMSCs results in sustained engraftment and myogenic differentiation,improves the postinfarction left ventricular(LV) perfusion and cardiac function.This research would provide experimental data for the treatment of ischemic heart disease with BMMSCs transferred with gene.Methods 1. BMMSCs were isolated from inbred line Wister rat's tibia and fibula with density gradient centrifugation,cultured in vitro,and induced with 5-azacytidine.The phase-contrast microscope,electron microscope, immunohistochemistry stain,and flow cytometry were applied to identify the induced cells.2.Our lab had constructed BMMSCs with Notch signal is activated without ligand through transfection of ICN through retrovirus-mediated gene transfer.We detect the express of ICN in BMMSCs after ICN transfection through Retroviridase polymerase chain reaction(RT-PCR).3.Acute myocardial infarction model of rats was constructed by occlusion of left coronary anterior descending branch.Chang of heart function was compared between post MI and before MI by echocardiogragm and electrocardiogram.4.80 inbred line Wister rats was random divided sham operation control group(A group,10 rats) and construction acute myocardial infarctionsurvived(AMI) group(70 rats),60 rats survival from operation were randomly and equally assigned to myocardial infarctionsurvived group(B group,15 rats),culture fluid transplantation group(C group,1 5 rats),BMMSC s activaed Notch signal transplantation group(D group,15 rats) and BMMSCs transplantation group(E group,15 rats).Cultuerd cells were labeled with DAPI 2 hours before cel ls transplantation.2 weeks after ligation of left coronary anterior descending branch,0.1ml volume of induced BMMSCs transferred with ICN,induced BMMSCs without transfection of ICN,culture fluid,and physiological saline were injected into the area of myocardial infarction,respectively.Echocardiography examinations were performed to evaluate heart function at day 1,28 after operation,respectively.Rats were sacrificed 28 days after operation,heart samples were fixed with formalin for pathologic examination. Morphologic change of heart samples were assessed by gross examination.Transplanted cells were located by fluorescence microscopy examination.Capillary density,which was identified by expression ofⅧfactor antibody,was counted in hearts,Desmin and troponinⅠantibody stain were used to identified the difeerntiation of transplanted cells.Expression of collagen and MMPs in rat's hearts was evaluated as well.Results 1.BMMSCs of rats was successfully segregated.Flow cytometry showed that cultured cells were positive for anti-CD29 antibody,anti-Flk-1 antibody,and anti-CD117 antibody.All of which confirmed that the induced BMMSCs differentiated into the myogenic cells.2.successfully constructed BMMSCs with Notch signal is activated without ligand through transfection of ICN through retrovirus-mediated gene transfer.We detect the express of ICN in BMMSCs after ICN transfection through RT-PCR.3.Myocardial infarction of rats was successfully constructed.ECG showed that ST segment was depressed and heart rates was faster when coronary artery was obstructed,ST segment was elevated and QRS complex was wider,and ST segment was depressed and Q wave appeared 8 hours later.Left ventricle was enlarged,local myocardial dyskinesis was found,and left ventricle Ejection Fraction(EF) decreased obviously by echocardiogram.Left ventricle EF of the operated rats(62.35±3.49)%, 24 hours after operation was lower than that of the rats before operation(39.67±2.33)%and sham operation rats(60.52±4.75)(p＜0.01).HE stain of heart samples collected 4 weeks after operation displayed local scar tissues devoid of cardiomyocytes,suggesting the formation of myocardial infarction.4.HE stain showed there was irregular scar tissues in anterior wall of left ventricle in cell D and E group,where allogenic cells(fluorescent cells) were found.Scar tissues but not allogenic cells were found in A group.Transplanted cells were positive for desmin and troponinⅠantibody.Echocardiogram demonstrated that local myocardial dyskinesis disappeared,volume of left ventricle got smaller,and left ventricle EF was elevated.Results of statistics analysis were as follows:heart function of rats in D group was beter than that of E group, B group,and C group.Infarcted areas and thickness of rats in D group were beter than that of E group,B group,and C group.Capillary density in D group was much more than that of others groups.Expression of type 1 collagen and MMP-1 in rats in induced D group was weaker than that of others groups.Conclunions Transplanted cells were alive in infarct myocardium and diferentiated into myogenic cells.Transplantation of BMMSCs transferred with ICN into myocardial infarction increased angiogenesis in infarct myocardium,decreased synthesis of collagen and matrix metalloproteinase,alleviated cardiac remodeling,and improved heart function.Transplantation of BMMSCs transferred with ICN was useful and safe for the treatment of myocardial infarction.