| Background:Myocardial infarction (MI) causes acute necrosis of cardiomyocytes. It is very dangerous and has a high rate of death. It is considered traditionally that cardiomyocytes can't regenerate after they are dead. Then Fibroblasts proliferate and repair the myocardium and the myocardium changes into scar tissue. It leads to left ventricular (LV) remodeling, which ultimately results in heart failure. Current therapeutic modalities including pharmacological treatment, percutaneous coronary intervention (PCI) and coronary artery bypass graft (CABG) mainly aim at improving the blood supply, but can't provide viable cardiomyocytes and can't well improve long-term prognosis of MI. Cell transplantation offers a new promise of rebuilding the damaged myocardium. Both experimental studies and clinical trials widely are carried out however the results of them are not consistent. It is not clear if the transplanted cells can differentiate into cardiomyocytes and if this therapy can improve one's heart function in the long term. The mechanism underlying this therapeutic effect is not fully understood and whether the cell transplantation causes arrhythmia is unclear.Objective:1.Left anterior descending coronary (LAD) artery ligation was used to produce AMI in hybrid canine.2.To explore the method of bone marrow mononuclear cell isolation and the feasibility of BM-MNCs being intracoronarily infused into infarction related artery.3. To investigate the effects of intracoronary autologous bone marrow mononuclear cells transplantation on cardiac function of AMI.4. To evaluate the labeling efficiency of CM-DiI and the lasting time of the fluorescence.5. To observe the survival and the differentiation of grafted bone marrow cells(BM-MNCs) in host myocardium.6. To abserve the effects of BM-MNCs transplantation on angiogenesis and the mRNA expression of cytokines such as VEGF,bFGF and MMP-9.7. To observe whether BM-MNCs transplantation can change the ERP of myocardium.8. To observe whether BM-MNCs transplantation can potentially cause arrhythmia.9. To observe whether the BM-MNCs transplantation can alter the spatial distribution of Connexins, which is important for arrhythmia genesis after MI.Methods:1.Sixteen hybrid canines were randomly divided into transplantation group(n=10) and control group(n=6). Left anterior descending coronary (LAD) artery ligation was used to produce AMI model.2. About 25-30ml of bone marrow was aspirated from the dogs' iliac bone of the transplantation group. Bone marrow mononuclear cell (BM-MNCs) suspension was prepared by density centrifugation using ficoll. The BM-MNCs labeled with CM-DiI which are observed in fluorescence microscope are prepared with the concentration of 3 X 107- 1 X 108/ml.3. BM-MNCs suspension was intracoronarily infused into infarction related artery 2 hour after AMI with needle and saline of the same volume was injected for the control dogs.4.We detected LVSP,LVEDP and used Swan-Ganz catheter to detect CO 2 hour after the ligation and 6 weeks after the BM-MNCs transplantation.5. To evaluate the heart function, we used echocardiography to detect LVESD,LVEDD,LVESV,LVEDV,FS,EF,SV 2 hour after the ligation and 6weeks after the BM-MNCs transplantation. 6. Canines are killed and histological examination was performed. The expression of Myosin heavy chain and Connexin 43 was assessed by immunofluorescence staining. Capillary density were assessed by vWF immunohistochemical staining; The mRNA levels of VEGF, bFGF and MMP-9 within infarct area were determined by RT-PCR 7. days after MI.7 The ERP of different areas in myocardium was assessed 6 weeks after cell transplantation, The expression of Connexin 43 was assessed by immunohistochemical staining.Results:1. The implanted cells showed clear membrane red fluorescence.2.6 weeks after the BM-MNCs transplantation, CM-Dil labeled BM-MNCs were mainly located within periinfarct and infarct area. Some BM-MNCs were positive for MHC and Cx43. Combined "CM-Dil and FITC" in images were observed.3. There isn't a significant difference of Hemodynamic indexs between 2 hours after the ligation and 6 weeks after the BM-MNCs transplantation in the control group (P>0.05). Compared to 2 hours after the ligation, LVEDP decreased significantly (5.1±3.07 vs 9.2±4.34, P<0.05) 6 weeks after MI in transplantation group. Compared to control group, LVEDP decreased significantly (5.1±3.07 vs 11.67±3.42, P<0.01) 6 weeks after MI. Compared to 2 hour after the ligation, CO increased significantly (3.1±0.89 vs 2.14±0.49, P<0.01) 6 weeks after MI in transplantation group. Compared to control group, CO increased significantly (3.1±0.89 vs 2.39±0.43, P<0.05)6 weeks after MI in transplantation group.4. There is not a significant difference of all the echocardiography index in control group between 2 hours after the ligation and 6 weeks after MI(P>0.05). Compared to 2 hours after the ligation, ESD,ESV,EDV,LVEF,FS,SV all improved in transplantation group 6 weeks after MI(P<0.05) and the same results exsists compared to the control group 6 weeks after MI(P<0.05). Compared control group, LVEF increased 7% in transplantation group. 5.Transplantation group increased the density of capillary significantly within periinfarct area 6 weeks after MI (19.32±2.47 vs 9.47±1.28, P<0.01), meanwhile within infarct area the density had no significant difference(3.44±0.51 vs 3.07±0.3, P>0.05).6. Transplantation group increase the mRNA level of VEGF 188,VEGF164 and bFGF significantly(1.16±0.38 vs 0.45±0.15, P<0.01) (1.17±0.3 vs 0.42±0.17, P<0.01) (0.90±0.21 vs 0.63±0.28, P<0.05) and decrease mRNA level of MMP-9 significantly (0.59±0.18 vs 0.93±0.3, P<0.05) 7. Compared control group, the ERP of infarct area,periinfarct area and normal area in transplantation group has no significant difference (85±9.26 vs 90±7.07ms, P>0.05) (87.78±9.37 vs 90±7.07, P>0.05) (85±11.95 vs 88.33±9.4, P>0.05). The expression of Cx43 in normal area is similar between transplantation group and control group(3543.7±446.00 vs 3431. 67±421.54, P>0.05). The expression of Cx43 in transplantation group in periinfarct area was significantly higer than that in control group((2312.50±412 vs 1356.17±332.73, P<0.05), but was still much less than in normal area(2312.50±412 vs 3543.7±446.00, P<0.05). The expression of Cx43 in infarct area is similar between transplantation group and control group (327.00±98.67 vs 311.33±78.74, P>0.05).Conclusions:1. The labeling efficiency of CM-DiI was more than 95% and the lasting time of the fluorescence in the host was more than 6 weeks. CM-DiI could be a good tracer for the BM-MNCs.2. The implanted BM-MNCs could survive in the infarcted lesion and differentiate into cells expressing MHC.and Cx43.3. The left ventricular systolic and diastolic function could be improved by BM-MNCs implantation.4. The BM-MNCs transplantation can increase capillary density, especially in the border zone of the myocardial infarction. BM-MNCs transplantation can increase the mRNA level of VEGF188,VEGF164 and bFGF, at the same time, it decreases the mRNA level of MMP-9.5. The spatial distribution of Cx43 gap junction was ameliorated by BM-MNCs transplantation, and it may ameliorate the arrhythmic susceptibility.
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