| Background:Heart failure is the final stage of various cardiovascular diseases. Nowadays, it is a major public health issue. Recently, cardiologists had made efforts to explore new ways of treating heart failure. Heart transplantation is an effective means, but some problems have restricted its developments, including donor sources, economic cost, ethical and so on. Implantation of cardiac assist device (LVAD) or CRT is invasive and expensive, can not be popularized. At the end of the last century, scientists found the adult stem cells could differentiate into other tissues. It is a new hope to resolve some difficult medical problems, which also includes the treatment of heart failure. The current study found that transplanted stem cells in vivo may not only differentiate into myocardial cells and vascular endothelial cells, also impact of myocardial micro-environment to improve cardiac function. However, the mechanism has not yet been fully clarified.Objective:1. To establish a heart failure model in dogs induced by right ventricular rapid pacing.2. To observe if BM-MNCs (labeled by CM DiI) can survive in the myocardium and differentiate into myocardial cells or vascular endothelial cells.3. To investigate BM-MNCs transplantation in improving cardiac function in heart failure mdoel in dogs.4. To evaluate the effect after transplantation of BM-MNCs on decreasing the apoptosis of myocardial cells.5. To observe SDF-1mRNA, CXCR4mRNA and AKT2mRNA expression in myocardium after BM-MNCs transplantation.Methods:1.16mongrel dogs were randomly divided into the graft group (n=8) and the control group (n=8). Each of dog was implanted a permanent pacemaker for rapid right ventricular pacing (230±10beats/min) for3to4weeks. The cardiac function was evaluated by echocardiography and hemodynamics parameters.2. The dogs’Bone marrow (25-30ml) was aspirated from anterior/posterior superior iliac spine under general anesthesia. BM-MNCs were isolated by Ficoll density separation on lymphocyte separation medium. After labeled by CM-Dil, BM-MNCs were counted with fluorescence microscope.3. CM-Dil labled BM-MNCs or saline were transplanted into myocardium via epicardium injection.4. Cardiac function was examined by hemodynamics and echocardiography4weeks later. The dogs were sacrificed with potassium chloride injection. The myocardium samples from left ventricular apex, left ventricular anterior wall and left ventricular septal were obtained to observe the distribution of the cells (labeled by CM-DiI) with fluorescence microscope.Immunofluorescence stain was performed to detect if a-sarcomeric actin expressed in the CM-DiI labeled cells.5. Cryosections were immunohistochemically stained using specific antibodies in PECAM-1(CD31) and SMA (a-smooth muscle actin) for counting the capillarys.6. The difference of Integral optical density (IOD) between the graft group and the control group were observed.7. Real-time quantitative PCR was performed to detect the expression of SDF-1mRNA, CXCR4mRNA and AKT2mRNA.Results:1. After3-4weeks of rapid pacing, the cardiac function of the dogs in both two groups significantly decreased in hemodynamic (RVP,17.38±2.06vs.16.08±2.78, P<0.01; PAP,19.08±2.10vs.15.54±2.26, P<0.01; PCWP,13.85±2.73vs.10.08±2.29, P<0.05; CO,2.55±0.54vs.3.50±1.05, P<0.05) and in ultrasound parameters (LVESD,4.35±0.42vs.3.72±0.40, P<0.01; LVEDD,4.92±0.34vs.4.34±0.46, P<0.01; LVESV,18.92±2.99vs.11.77±3.52, P<0.01; LVEDV,29.85±3.87vs.26.92±4.03, P<0.01; SV,9.92±2.36vs.15.31±4.09, P<0.01; EF,36.78±5.92vs.65.38±7.08, P<0.01; LVPWd,5.38±1.27vs.6.72±1.14, P<0.01).2. The CM Dil labeled cells with red fluorescence were found four weeks after cell transplantation. A green fluorescence was demonstrated by the a-sarcomeric actin immunofluorescence stain. Some cells stacked with the CM Dil labeled cells in the graft group were found a yellow fluorescence. Only green fluorescence was seen in the control group.3. The hemodynamic and echocardiographic parameters significantly improved in the graft group compared with the control group four weeks after cells transplantation (LVEF,42.14±2.79vs.37.29±6.75, P<0.05; SV,13.28±1.80vs.9.43±1.90, P<0.01; LVPWd,6.12±1.43vs.5.54±1.02, P<0.01; PAP,16.29±1.90vs.19.86±2.34, P<0.05; PCWP,12.29±1.89vs.14.71±3.15, P<0.05; CO,2.95±0.40vs.2.66±0.47, P<0.05).4. The capillary density was significantly higher in the graft group than that in the control group (P<0.01).5. The IOD in the graft group was higher than that in the control group (322.46±91.03vs.130.40±34.22, P<0.01).6. The mRNA levels of SDF-1, CXCR4and AKT2were higher in the graft group than those in the control group (SDF-1,1.09±0.65vs.0.43±0.15, P<0.01; CXCR4,1.88±2.63vs.0.39±0.24, P<0.01; AKT2,1.10±0.56vs.0.64±0.15, P<0.01).Conclusion:1. These results indicated that the BM-MNCs transplanted into myocardium by epicardium injection could survive and differentiate into cardiomyocyte-like cells.2. BM-MNCs transplantation promotes angiogenesis.3. Autologous BM-MNCs transplantation improves cardiac function.4. Autologous BM-MNCs transplantation improves the mRNA level in SDF-1, CXCR4and AKT2. |
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