| ObjectivesTo evaluate the stability and reliability of dog chronic stem cell myocardium transplantation models produced by ligating the left anterior descending coronary artery or by cryoinjuring myocardium with ~18F-FDG position emission tomography (PET) and ~99TC~m-MIBI single photon emission computed tomography (SPECT).Dog bone marrow CD34~+ cells were separated and purified with immunomagnetic separation methods and implanted into the chronic cryoinjured myocardium site. To evaluate the changes of perfusion with ~99Tc~m-MIBI and metabolism with ~18F-FDG in refrigerant infarction myocardium implanted autologous bone marrow CD34~+ cells. The expression of basic fibroblast growth factor (bFGF), blood vessel endothelium growth factor (VEGF) , factor VIII and dismin in refrigerant infarction myocardium implanted bone marrow CD34~+ cells were detected by immunohistiologicalmethod, and the density of vascular was calculated, in order to evaluate the revascularization of infarction myocardium implanted autologous bone marrow CD34~+ cells. To oberseve the changes of myocardium fiberous in infarction myocardium befor and after cell transplantation and search myocardium juvenilecells by pathology.A method to enrich ideal human bone marrow CD34~+ cells assoon as possible from breast bone and costal bone during coronary artery bypass graft (CABG) was explored for clinical application smoothly.MethodsTwelve dogs were randomly divided into deligation and cryoinjuring group, in which chronic myocardial infarction model was produced by ligating the left anterior descending coronary artery or by cryoinjuring myocardium. Myocardial perfusion imaging was evaluated by 99TCm-MIBI single photon emission computed tomography (SPECT) and metabolic imagingwas evaluated by ~18F-FDG position emission tomography (PET) before and 4, 8 weeks later operation. F values of reduced myocardial segments and matching of myocardial perfusion and FDG uptake were calculated.Thirty-six dogs were randomly divided into cell transplantation and control group. In cell transplantation group bone marrow CD34+ cells were separated and purified with immunomagnetic separation methods and implanted into the chronic cryoinjured myocardium site, and in control group IMDM culture fluid was injected into the chronic cryoinjured myocardium site. Myocardial perfusion imaging was evaluatedby 99Tcm-MIBI single photon emission computed tomography and metabolic imaging was evaluated by 18F-FDG positron emission tomography before and 4 weeks after myocardium infarction and 8 weeks after cells transplantation. Every detecting head harvested 60 original images original those matrix 64><64. Level long axis, vertical long axis and minor axis imagings were made by Butterworth filtered back projection. F values of reduced myocardial segments of myocardial perfusion and FDG uptake were calculated. The specimens in infarction myocardium site were harvested at 2, 4, 8 weeks later for the density of vascular, expression of bFGF, VEGF, factor VI and dismin examination by immunohistiological method. Gray scale value was reflected the expression calculated by KS400 image analysis system. Pathological examination was made to obseve the cell shap, search myocardium juvenile cells or abnormal cells by light andelectron microscope.During coronary artery bypass graft, several stab sites werechosen from breast bone and costal bone of patient, and 20~ 30ml human bone marrow was collected. These bone marrows were isolated and purified by mean of immunomagnetic beads on MiniMACS device. The purity, cell vitality and quantity of bone marrow CD34+ cells detected by flow cytometry and light microscope after purification.ResultsThere was no death in all chronic myocardial infarction models. F values of myocardial perfusion were 10.47±0.51, 6.67±1.03, and FDG uptake 10.33±0.66, 5.83±0.75 in deligation group(p<0.05), F values of myocardial perfusion were5.67±0.82> 5.17±0.98, and FDG uptake 5.5O±1.31, 5.00±1.55 in cryoinjuring group (p>0.05) at 4, 8 weeks later operation. In the reduced myocardial segments, infarct myocardium was 41.9% and 70% at 4, 8 weeks later operation in deligation group, 91.7% and 100% in cryoinjuring group.The images of 99Tcm-MIBI and 18F-FDG in normal myocardium were clear. F values of myocardial perfusion were 1.17±0.41, 1.50±0.55 and FDG uptake 5.5O±1.31, 5.44±0.70 in cell transplantation group(p<0.01) before and after 8 weeks transplantation, 5.00±l.55, 5.08±1.46 and 5.53±0.80, 5.54±1.29 in control group (p>0.05). In transplantation group, the perfusion and metabolism imaging was low before transplantation, but recover after 8 weeks transplantation significantly. There was not market change in control group. Autologous bone marrow CD34+ cells could obviously improve the vascularization and coronary perfusion and recover the glycometabolism which showed that there were many living myocardial cells in the chronic cryoinjured myocardium site.Detected by factor VI immunohistiological method, the vascular density was 3.2±1.2, 8.2±0.8, 12.7±2.2 at the implant site in 2nd, 4th, 8th week after transplantation, higher than that of in the control group(p<0.05). The expression of bFGF and VEGF in normal myocardium was stable. The gray scale value of bFGF and VEGF in cell transplantation site at 2, 4, 8 weeks later was 85.73±3.17, 70.39±2.88, 73.72±4.23 and 90.24±5.03, 81.28±3.67, 78.12±4.66, higher than of normal myocardiium 55.48±2.63, 54.46±2.78, 53.99±3.14 and 51.17±2.24, 50.67±2.38, 52.06±3.22 ( p<0.05 ) ,and higher than that of infarct myocardium in controlgroup 50.02±2.41, 55.46±4.36, 60.38±5.03 and 53.37±2.61, 60.72±3.52, 64.29±4.01(p<0.05).There were angiogenesis factors in infarction myocardium implanted bone marrow CD34+ stem cells.The expression of dismin in infarct myocardium was intensive, it's gray scale value at 2, 4, 8 weeks later transplantation was 84.41±4.65, 98.37±5.01, 90.24±3.95, higher than of normal myocardiium 68.24±2.91, 70.55±4.40, 68.98±4.54 ( p<0.05 ) . The expression of dismin in CD34+ cells transplantation site was descending, it's gray scale value at 4, 8 weeks later transplantation was 73.51±4.92, 72.63±5.02, lower than of control group(p<0.05) and no difference significently than that of infarct myocardium(p>0.05). There were a myocardium muscularizing proceding in infarction myocardium implanted marrow bone CD34+ stem cells.Myocardium was necrotic and fiberize in infarction myocardium, there was no normal cells. But many normal myocardiums myocardium juvenile cells were found in infarctionmyocardium implanted CD34+ cells by pathology examination. During coronary artery bypass graft, 20~30ml human bonemarrow was collected from breast bone and costal bone of patient. These bone marrows were isolated and purified by mean of immunomagnetic beads on MiniMACS device within 3.5-4 hours. The purity, cell vitality and quantity of bone marrow CD34+ cells detected by flow cytometry after purification was 93.7%~98.3% mean 95.8±1.6% , 92.1%~97.8% mean 95.3±1.6% and 0.78xl07~3.6xl07 mean (1.7±0.8)xl07 respectively. These procedures had been done end of CAGB.
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