The Experimental And Clinical Study Of The Bone Marrow Mononuclear Cells Transplantation In Chronic Heart Failure

In recent decades, several therapeutic options have been developed or improved to delay the progression of ventricular dysfunction in patients with heart failure. However, plain reversion of the process has not yet been obtained, and the prognosis of these patients remains very limited. Given the limited efficacy of the current pharmacological therapeutic alternatives for the treatment of heart failure, other forms of treatment have been developed.The demonstration of the capacity of adult bone marrow cells to differentiate in vitro into various cell types was the initial stimulus for their experimental use in the treatment of heart failure. Strauer et al were the first to demonstrate the feasibility and safety of intracoronary infusion of bone marrow mononuclear cells(BMMNCs) for the treatment of patients with ischemic cardiomyopathy. Within the past few years, researchers have made many advances in their understanding of BMMNCsand have described beneficial effects of transplantation of autologous mononuclear bone marrow in patients with chronic postinfarction heart failure. Recent publications have found intracoronary BMMNCs transplantation is a feasible and safe therapeutic strategy to improve symptoms, decrease the brain natriuretic peptide and atrial natriuretic peptide serum levels, increase cardiac function, and possibly prolong life in patients with end-stage heart failure. Cell therapy has emerged as a promising option for the treatment of advanced cases of heart failure.However, there is much controversy regarding stem cell biology and cardiac regeneration, and some investigators denied that cell transplantation increases heart function. As the actual mechanisms of cell transplantation-induced functional improvement in heart failure, it is even elusive. Our study had confirmed the value of the minor myocardial damage in CHF.In this study, we prospectively investigated the safety and effects of BMMNCs transplantation and a secondarily ddresses the hypothesis that transplantation of BMMNCs could promote neovascularization and may decrease myocardial damage in CHF. At last, we discuss the mechanism of the transplantation of BMMNCs to overcome the failure of the natural myocardial healing process.The present study primarily include two part: the experimental and clinical study. The experimental study include three part: 1)Isolation culture, and proliferation of rats bone marrow mesenchymal stem cells in vitro; 2)The experimental study of the chronic heart failure model after myocardial infarction in rats; 3)The study of effects of exogenous bone marrow mononuclear cell transplantation on cardiac function in chronic heart failure rats after myocardial infarction. The clinical study was clinical research of autologous mononuclear bone marrow cells transplantation in patients with chronic ischemic heart failure.Part oneISOLATION CULTURE, AND PROLIFERATION OF RATS BONE MARROW MESENCHYMAL STEM CELLS IN VITROObjective To investigate the optimal condition for isolation, culture and proliferation of bone marrow mesenchymal stem cells(BMMSCs), and to observe the growth and biology characterization of BMMSCs, and to provide a seed cell for cell transplant to cure all kinds of organ failure.Methods Bone marrow was collected from 2-month old Wistar rat by flushing femurs and tibias under sterile condition. BMMNCs were isolated from those bone marrow using density gradient centrifugation, and were cultured in low-glucose DMEM supplement with 15% FBS for expanding. When covering the flask, the cells were passaged after which were digested by pancreatin, and were observed their morphologic changes under inverted microscope.Results Primary cultured MSCs adhered to plastic surface within 72 hours and formed the colonies of BMMSCs at 4^th day. From 5th to 12th day, BMMSCs grew faster and got together to form bigger colonies then before. After 14 days from the beginning, the BMMSCs fused in monolayer; then were subcultured by 0.25% trypsine-0.02%EDTA.The passage BMMSCs proliferated fast, and could be subculture about every 5 days. In later generation, BMMSCs grew low, and mutlipled karyotype appeared.Conclusion BMMNCs which contained many BMMSCs can be obtained via density gradient centrifugation. The BMMSCs could be isolated, cultured , expanded and purified easily in vitro, so it can be used as a method to gain the seed cell for curing organ failure.PART TWOTHE EXPERIMENTAL STUDY OF THE CHRONIC HEART FAILURE MODEL AFTER MYOCARDIAL INFARCTION IN RATSObjective To establish and assess the rat model of chronic heart failure(CHF) after myocardial infarction, to study the hemodynamic character and the change of the echocardiography and electrocardiography after MI in rat, and to prepare mesenchymal stem cells for CHF treatment with cell transplantation.Methods Rats were anesthetized by intraperitoneal injection of pentobarbital sodium solution and a thoracotomy was performed and the left anterial descend artery was ligated to cause myocardial infarction , without intubated and ventilated, along the middle of the sternum with pleura cavity integrity. Then the cause of death, the change of echocardiography, electrocardiography and hemodynamic parameters, and area of the infarction were observed and measured in eight weeks. Sham-operated rats and blank control rats were served as controls. Results1) Of the 30 rats that underwent LAD ligaration, 7 died in surgery, primarily from hemorrhage and arrhythmia. Of the 23 remaining rats, 4 died within 8 week of myocardial infarction, primarily from heart failure. The perioperative mortality was 36.67% within 8 week.2) The ST segments on I,aVL,II,III,aVF leads elevated to 0.2mV persistently after ligation of LAD and the pathological Q-wave formed after 24 hours in I,aVL leads.3) There were no difference before operation as for echocardiography parameters(P>0.05). After eight weeks, compare with sham-operated group and control group, in the operated group, the Left ventricular anterior wall showed hypokinesis, akinesis or dyskinesis. The end systolic interventricular septal thickness [(1.58±0.34 vs 2.16±0.16 vs 2.08±0.19)mm ,P all< 0.05], the end diastolic interventricular septal thickness[(1.42±0.30 vs 1.89±0.21 vsl.81±0.24)mm ,P all< 0.05],the Left ventricular end-systolic posterior wall thickness[ (2.05±0.39 vs 2.71±0.12 vs 2.60±0.16) mm ,P all<0.05], the Left ventricular end- diastolic posterior wall thickness[ (1.84±0.34 vs 2.47±0.11 vs 2.33±0.11) mm ,P all<0.05],the Left ventricular end-systolic diameter[ (5.22±1.07 vs 3.70±0.58 vs 3.81±0.44) mm ,P all<0.05] and the Left ventricular end-diastolic diameter[ (7.05±1.03 vs 5.63±0.58 vs 5.68±0.45) mm ,P all<0.05] increased significantly. The Left ventricular ejection fraction[ (44.47±13.87 vs 80.00±3.86 vs 79.00±3.43) %,P all<0.01] and fractional shortening[ (19.47±7.66 vs 42.60±4.58 vs 41.67±3.57) %,P all<0.01] decreased significantly. There were no difference between sham-operated group and control group(P>0.05).4) at 8^th week, compare with sham-operated group and control group, the left ventricular systolic pressure[(l23.42±13.20 vs 142.70±l 1.10 vs 146.1 l±14.74)mmHg, P<0.01], the absolute value of +dp/dt_max [(4760.16±634.25 vs 6674.20±628.57 vs 6622.88±542.04)mmHg/s, P < 0.01]and - dp/dt_max [(4543.89±628.14 vs 6439.40±612.67 vs 6381.44±539.38)mmHg/s, P<0.01] decreased significantly. The left ventricular end diastolic pressure increased[(15.13±3.83 vs 3.40±0.81 vs 3.68±0.65)mmHg, P < 0.01]significantly. There were no difference between sham-operated group and control group(P>0.05). and decreased in model group.5) at 8^th week, there were 9 rats whose LVEF within 45%-70% and were defined left ventricular dysfunction, and there were 10 rats whose LVEF under 45% were defined heart failure. The LVEDP exceeded 15mmHg in rats with heart failure.6) Pathology examination in coronary ligation group showed that in infracted area cardiomyocytes were replaced by fibrous tissues with few subendocardial cardiomyocytes, The total infracted area ranged from 19% to 43.20%.Conclusion1) the large area myocardial infarction could be formed after the proximal segment of anterior descending coronary artery was ligated and the chronic heart failure model can be established in 8 weeks in wistar rat.2) The persistent ST-segments elevation(0.2mV) on I,aVL,II,III,aVF leads of electrocardigram can be as the early index of the large area myocardial infarction and the the pathological Q-wave on I,aVL leads of electrocardigram can be as the index of MI.3) The echocardiography suggested that the operated groups had significantly more dilated left ventricles with lower EF and FS than those of the control group. The rat model of chronic heart failure could be established by EF whose value was under 45%. It is important to study the chronic heart failure.PART THREETHE STUDY OF EFFECTS OF EXOGENOUS BONE MARROW MONONUCLEAR CELL TRANSPLANTATION ON CARDIAC FUNCTION IN CHRONIC HEART FAILURE RATS AFTER MYOCARDIAL INFARCTION.Objective: To explore the long-term effects of bone marrow mononuclear cells transplantation on cardiac function and its mechanism in chronic heart failure rat. Methods: First, to establish 30 chronic heart failure rat model(inbreeding line) which were divided into 3groups at random: blank(group A, n=10),control(group B,n=10) and transplantion (group C,n=10). BMMNCs isolated from the same inbreeding rats were directly injected into the myocardium of transplantation group, control group were injected culture solution, and sham-operated rats were underwent the surgery but not transplantation. Eight weeks after BMMNCs transplantation, echocardiography and hemodynamics examination were performed in all groups to observe the left venticular ejection fraction (LVEF) , fractional shortening(FS), end systolic interventricular septal thickness(IVSTs), the end diastolic interventricular septal thickness(IVSTd), the Left ventricular end-systolic posterior wall thickness(LVPWs) , the Left ventricular end- diastolic posterior wall thickness (LVPWd), the Left ventricular end-systolic diameter(LVEDs) and the Left ventricular end-diastolic diameter(LVEDd) for evaluation of the cardiac function and left ventricular remodeling. The changes of left ventricular systolic pressure (LVSP),left ventricular end-diastolic pressure(LVEDP) and the maximal ratio of Left ventricular diastolic pressure to left ventricular diastolic time(±dp/dtmax)were also measured for evaluation of hemodynamic changes. Fluorescence microscope was used to detect cells labeled with DAPI. The number of vessels and the vasculogenesis in the infracted area were examined by histology study. TUNEL method was used to analyse cardiac myocyte apoptosis in the area far away from ischemic area.Result1)The DAPI-labeling ratio of rats BMMNCs was 100%,and labeled cells could survive well in vitro.2)There were no difference before cell transplantation as for echocardiography parameters in 3 groups(P>0.05). After eight weeks of cell transplantation, compare with black group and culture solution group, in the cell transplantation group, LVEF[(45.90±8.05 vs 32.43±4.35 vs 32.11±4.26), P<0.01], FS[(17.40±4.31 vs 12.84±4.56 vs 12.97±4.37) , P < 0.05], IVSTs[(1.67±0.24 vs 1.39±0.17 vs 1.38±0.12)mm,P<0.05], IVSTd[ (1.53±0.21 vs 1.32±0.13 vs 1.32±0.10) mm, P< 0.05], LVPWs[ ( 1.84±0.21 vs 1.59±0.20 vs 1.54±0.16) mm, P<0.05] and LVPWd[(1.66±0.16 vs 1.46±0.16 vs 1.43±0.12) mm, P<0.05] increased significantly. The LVEDs[ ( 5.95±0.86 vs 7.48±1.28 vs 7.48±1.14 ) mm, P < 0.05] and LVEDd[(7.19±0.85 vs 8.53±1.12 vs 8.55±0.96)mm, P<0.05] decreased significantly. There were no difference between sham-operated group and control group(P>0.05).3)at 8^th week, compare with black group and culture solution group, in the cell transplantation group, LVSP[ ( 141.80±16.63 vs 122.13±12.56 vs 124.00±12.93)mmHg, P < 0.05], the + dp/dt_max [ ( 5362.00±807.99 vs 4406.25±432.47 vs 4532.22±411.76 ) mmHg/s, P < 0.05] and - dp/dt_max [ (4897.00±823.81 vs 4406.25±432.47 vs 4135.56±351.32, P<0.05) mmHg/s, P< 0.05] increased significantly. The LVEDP[(10.89±2.62 vs 18.22±2.47 vs 17.16±2.49)mmHg, /><0.05] decreased significantly. There were no difference between black group and culture solution group (P>0.05).4)No DAPI labeled cell was found in all slices.5)Local capillary density in infarcted area was much higher in group of transplantation of BMMNCs than in black control group and PBS group (2.14±0.62) VS (2.37±0.74) VS (7.08±1.06) ,each P<0.01,but there was no significant difference between control group and PBS group ,P>0.05.6)Apoptosis ratio in nonischemic area in group of transplantation of BMMNCs was much lower than the other two groups(2.61±1.134 vs 72±1.13 vs 4.54±1.21, P< 0.01, each P<0.01),and no difference between black control group and PBS group, P=0.94.Conclusions1)BMMNCs transplantation can improve the cardiac function in chronic heart failure rats; a little PBS transplantation has little or no effect on the cardiac function in CHF rats.2)There is little or no relationship between the improvement of heart function and transdifferentiation of transplanted BMMNCs in CHF rats.3)BMMNCs transplantation could increases local capillary density in infarcted area and decrease cardiac myocyte apoptosis, these may help to improve the heart function in rats 8 weeks post-infarction. PART FOURCLINICAL RESEARCH OF AUTOLOGOUS MONONUCLEAR BONE MARROW CELLS TRANSPLANTATION IN PATIENTS WITH CHRONIC HEART FAILUREobjective to research the feasibility and efficacy of autologous bone marrow mononuclear cells (BMMNCs) transplantation on the improvement of cardiac function and the serum level of cardiac troponin I(cTnI) in patients with chronic heart failure(CHF).Methods Thirty patients with CHF treated with percutaneous coronary intervention (PCI) were randomized in a 1:1 way to either intracoronary transplantation of autologous BMMNCs(n=15) right after PCI or sodium chloride concluding heparin(controlled,n=15) via a over-the-wire(OTW) balloon catheter slowly placed into the infarct-related artery during balloon dilatation. The Left ventricular end-diastolic diameter(LVEDD), The left venticular ejection fraction (LVEF) and the ratio of E to A (E/A) were determined by color Doppler echocardiography and the serum cTnI were measured before and 6 months after the procedure in all patients.Results1)There were no significant differences in the mean NHYA class, risk factors of CAD and the severity of coronary artery desease among the two randomized groups(P >0.05), and there were also no significant differences in treatment aspect beside transplantation of autologous BMMNCs. All patients' bone marrow were collected and transplanted successfully, the number of bone marrow cells transplanted were( 9.25±1.18)×10⁷BMMNCs and there were no major periprocedural complications include ventricular contraction and myocardial ischaemia. 2) After six months of follow up, the symptoms and cardiac function were significantly improved in the cells transplantion group. LVEF(32.13±4.52 vs 40.20±5.19, P<0.01) and E/A(0.89±0.09 vs 0.96±0.10, P<0.01) significantly increased , and NHYA class (3.67±0.49 vs 2.47±0.64, P<0.01) and LVEDD [(69.01±8.62 vs 65.22±8.28)mm, P<0.05]decreased significantly. In the control group, cardiac function also improved, but not significantly. After six months of follow up, compared with control group, the LVEFC 33.53±4.81 vs 40.20±5.19, P <0.01) and E/A(0.87±0.07 vs 0.96±0.10, P<0.01) significantly increased , and NHYA class (3.23±0.70 vs 2.47±0.64, P<0.01) and LVEDD (71.43±8.21 vs 65.22±8.28, P<0.05) decreased significantly in cells transplantation group.3) Serum cTnI lever decreased significantly than control group[(0.071±0.050 vs 0.037±0.038)ng/ml, P<0.05], and also decreased significantly than before cells transplantation [(0.074±0.032 vs 0.037±0.038)ng/ml, P<0.05].Conclusion Intracoronary transplantation of autologus BMMNCs can decrease the serum cTnI lever and can also improve their systolic and diastolic function at the same time in patients with old myocardial infarction. In spite of the many challenges that remain, the autologous BMMNCs transplantation is a brand-new biological-therapy, which would offer a fresh approach for heart failure.