Comparison Of Bone Marrow Mesenchymal Stem Cells Transplantation And Mobilization Of Bone Marrow Stem Cells For The Therapy Of Chronic Heart Failure

Despite the advances made in the management of acute myocardial infarction (AMI),chronic heart failure(CHF)secondary to left ventricular remodeling following infarction continues to be a major medical problem world-wide After myocardial infarction,the infarct area is replaced by fibrocytes and collagen fibers.The nonelastic scar eventually thins and dilates,thereby causes CHF.Several therapeutic options have been developed quickly to delay the progression of ventricular dysfunction in patients with AMI.However,there are more than 30%patients with successful revascularization causes left ventricular remodeling and CHF.Patients with CHF have a poor prognosis.Emerging research in cardiac therapy is destined to regenerate infracted myocardium and enhance the rate of survival in cardiac patients.Many experimental studies have shown that bone marrow stem cells are capable of regenerating infracted myocardium and inducing myogenesis and angiogenesis; this leads in turn to amelioration of cardiac function in rats.Bone marrow mesenchymal stem cells(BMMSCs)transplantation and bone marrow stem cells mobilization with granulocyte- colony stimulating factor(G- CSF) are two strategies for the repair of infraeted myocardium which have been used in clinical study.Stem cells are undifferentiated immature precursor cells that can proliferate, self-renew,and differentiate into one or more types of specialized cells,including cardiomyocytes.Animal studies have revealed that autologous BMMSCs transplantation can repair the infracted myocardium and improve cardiac function.The present study demonstrated that G- CSF could mobilize bone marrow stem cells home to the infracted myocardium.Orlic reported that bone marrow stem cells mobilized to the mice damaged myocardium differented to myocytes, endothelial cells and smooth muscle cells.But Norol found that,in a baboons model, only endothelial cells were observed in the infracted area,without detectable differented myocytes and smooth muscle cells.G- CSF had been reported to accelerate the healing process by promoting the absorption of necrotic tissues via increase of macrophages and reduces granulation and scar tissues for the beneficial effect of CHF treatment.For the therapy of CHF,the effective cell component of bone marrow mobilization with G- CSF is the same as BMMSCs transplantation.However,the comparison between these two methods remains largely uninvestigated.We aimed to analyze the action mechanism and effect of BMMSCs transplantation on cardiac function after myocardial infarction.we compare them to explore more practical and effectively stem cells therapeutic strategy for CHF.The study include three part:(1)The experimentalstudy of the chronic heart failure model after myocardial infarction in rats.(2)Isolation,culture and proliferation of bone marrow mesenchymal stem cells in vitro.(3)Comparison of bone marrow mesenchymal stem cells transplantation with bone marrow stem cells mobilization for the therapy of chronic heart failure in rats.PART ONE THE EXPERIMENTAL STUDY OF THE CHRONIC HEART FAILURE MODEL AFTER MYOCARDIAL INFACTION IN RATSAbjective It is one of critical techniques for medical experiment to make myocardial infarction-heart failure model.The Purpose of this study is to establish and assess the model of chronic heart failure(CHF) after myocardial infaction(MI) in male Wistar rats.Methods Left anterior descending coronary artery(LAD) was ligated to induce myocardial infarction in male Wistar rats.Electrocardiograms were recorded.The control group(n=10) was as same as MI group excep for ligating LAD.Echocardiography was recorded in all experimental subjects before and after operation,using a 10-MHz phased-array transducer and SONOS 7500 echocardiography system.M-mode tracing and 2D echocardiography images were recorded from the parasternal long- and short-axis views.Left ventricular end-systolic dimensions(LVDs) and end-diastolic dimensions(LVDd),systolic and diastolic anterior wall thickness(LVATs,LVATd),as well as systolic and diastolic interventricular septal thickness(IVSTs,IVSTd)were measured from the M-mode tracings.For each M-mode measurement,at least three consecutive cardiac cycles were sampled.LV ejection fraction(LVEF) and fractional shortening(FS) were derived from LV cross-sectional area in 2D short-axis view.Standard formulae were used for echocardiographic calculations.All data were analyzed offline with software installed in the ultrasound system.All measured and calculated indexes were presented as the average of three consecutive measurements.Hemodynamic parameters including heart rate,left ventricular systolic pressure (LVSP),left ventricular end diastolic pressure(LVEDP),maximal velocity increase of pressure(+dp/dtmax) and maximal velocity decrease of pressure(-dp/dtmax) were observed.Rats in both groups were sacrificed and the histopathologic examinations were performed 8 weeks later.Results(1)The ECG ST-segments onâ… ,â…¡,â…¢,aVR,aVL,aVF leads elevated to 0.2mv persistently after ligation of left anterior descending coronary artery.After 1-3 days, the presence of Q-waves on ECG tracings indicated that myocardial infarction was induced successfully in rats.(2) Echocardiography was found that the wall thickness and motion of the LV anterior wall in model group were thinned and akinetic 8 weeks after MI.In the model group,LVDs,LVDd increased significantly(P＜0.05);LVATs,LVATd,IVSTs,IVSTd group decreased significantly(P＜0.05);FS[(29.56±7.82)vs(42.57±5.43),(P＜0.01)],LVEF[(40.68±13.76%)vs(80.15±4.79%),(P＜0.01)]decreased significantly.(3) At 8th week,absolute value of + dp/dtmax and - dp/dtmax in model group were lower than those in control group(P＜0.05).LVEDP were higher than those in control group.(P＜0.01).(4) Histopathologic examinations showed there were massive inflammatory cell infiltration and fiber tissue proliferation in infracted area myocardium.The total infracted area are ranged from 19%to 42.3%.Conclusions The heart failure model forms at 8 week afterM I.The survival rate can improve with proper measures taken during and after the operation.This method can ideally induce congestive heart failure model.It is of important significance for the research of heart failure.PART TWO ISOLATION CULTURE AND PROLIFERATION OF BONE MARROW MESENCHYMAL STEM CELLS IN VITROAbjective The purpose of the study are to establish a simple,rapid,practical method and to prepare the optimal condition for the in vitro isolation,culture,purification and proliferation of rat bone marrow mesenchymal stem cell(BMMSCs).To observe the growth and biology characterization of BMMSCs,and to provide seed cells for the therapy of CHF.Methods.Rat BMMSCs were isolated and purified by differential adhesion method. The femora bone marrow was distilled by acentric means and the monomuclear cell was isolated by lmphocyte parting liquid,then the third generation of bone marrow cells were obtained through the process of swashing,culturing and generating.The BMMSCs were incubated with 4,6-diamidino-2-phenylindole(DAPI) which density is 50ug/ml and swashed by Hanks liquid,so it labeled by the fluorescence Dye.The karyons which emit blue fluorescence in the same visual field was observed by fluorescence microscope and The labelled ratio was obtained through counting the karyons' average.Results Primary cultured BMMSCs adhere to plastic surface within 72hours and formed the colonies of BMMSCs at 4th day.From 5th to12th day,BMMSCs grew faster and got together to form bigger colonies then before.After 14 days,the BMMSCs fused in monolayer,then subcultured by 0.25%typsine.The passage BMMSCs proliferated fast,and could be subculture about every 5 days.The third generation of bone marrow cell(P₃) was obtained.In later generation,BMMSCs grew low and multipled karyotype appeared.Rat BMMSCs were effectively purified by differential adhesion method,and grew well with good proliferation and normal morphology in medium.BMMSCs was labelled with DAPI and the labelled ratio was＞97%.Conclusions(1) Bone marrow stem cells which contained many BMMSCs can be obtained via density gradient centrifugation.(2) The BMMSCs could be isolated,cultured,expanded and purified esaily in vitro.DAPI labelling is sensitive and highly efficient,so it can be used as a method to labelling cells.(3) BMMSCs can be used as a method to gain seed cells for the therapy of chronic heart failure. PART THREE COMPARISON OF BONE MARROW MESENCHYMAL STEM CELLS TRANSPLANTATION WITH BONE MARROW STEM CELLS MOBILIZATION FOR THE THERAPY OF CHRONIC HEART FAILURE IN RATSObjective To compare bone marrow mesenchymal stem cells(BMMSCs) transplantation with bone marrow stem cells mobilized by granulocyte-colony stimulating factor(G-CSF) for the therapy of chronic heart failure(CHF) in rats,and to explore more effective and practical stem cell therapeutic strategy for CHF.Methods Myocardial infarction of left ventricle was induced in rats by ligation of left coronary artery.The rats were randomly divided into transplanted group, mobilized group and control group.In transplantation group(n=10),BMMSCs transplantation was performed after CHF.BMMSCs labelled with DAPI were transplanted into the infarcted myocardium through intramyocardial injection 4 week later.In mobilization group(n=10),G-CSF(30μg/kg·d) was injected subcutaneously after CHF and every 24 hours for 5 days.Control animals(n=10) did not receive any treatment after CHF.Echocardiography were performed for the evaluation of cardiac function 4 weeks later.Hemodynamic parameters including left ventricular systolic pressure(LVSP), left ventricular end diastolic pressure(LVEDP),maximal velocity increase of pressure per second(+ dp/dtmax) and maximal velocity decrease of pressure(-dp /dtmax) were observed.Histological examination and immunohistochemical analyses were performed to detect BMMSCs's proliferation,differentiation and their angiogenic function 4 weeks later.The infarct size and vascular density in infarct zones were measured accordingly. The expression levels of Bcl-2,Bax and the apoptosis- associated genes were analyzed semi-quantitatively by RT-PCR.Results(1) Four weeks after cell transplantation,theBMMSCs labelled DAPI were not found in necrosis zone in transplanted group,but there were a great deal of neovascularization in necrosis zone.No regeneration of smooth muscle cells and cardiomyocytes were found in the infarcted area.(2) Echocardiography indicated that LVEF decreased significantly in control group 4 weeks later.In transplanted and mobilized group,the heart function had a great improvement and LVEF increased significantly than that in control group(P＜0.05),and there was no significant difference between the transplanted and mobilized group(P＞0.05).(3) Mobilized group and transplanted group had higher LV +dp/ d t max and-d p/d t max,lower LV end - diastolic pressure compared with group control group 4 weeks later(P＜0.05).(4) Histological studies revealed that the vascular density of mobilized group and transplanted group in the infarcted area were significantly greater in comparison with control group[(6.58±1.47) vs(7.06±1.63)vs(2.14±0.62), (P＜0.01)].(5) Both G-CSF and BMMSCs transplantation can reduce the numbers of the cardiomyocyte apoptosis(P＜0.05),and there was no significant difference between the transplanted and mobilized group(P＞0.05).(6) In the transplanted and mobilized group,Bcl- mRNA expression increased markedly(P＜0.05),but Bax mRNA expression decreased evidently(P＜0.05) compared to those in the control group.Conclusions(1)This experimental study indicated that both G-CSF and BMMSCs transplantation can promote angiogenesis,reduce the numbers of the cardiomyocyte apoptosis in the infracted area and can significantly improve the heart function for the therapy of CHF.(2)Bone marrow stem cell mobilization may offer a new and non-invasive therapeutic strategy for CHF.