| Obiectives To compare the repair effects of different routes of transplantation of bone marrow mesenchymal stem cells on acute myocardial infarction in rats, and to explore a better way to transplant.Methods 1 Purification of cells by whole bone marrow adherence screening method.P3 cells were grown on behalf of the cells, and the methods of immunocytochemistry were detected, immunocytochemistry for identification by the expression of cells surface marker CD34, CD44; 2 5-azacytidine induced differentiation of bone marrow mesenchymal stem cells into cardiomyocyte like cells by induction, and then the cells were identified by immunocytochemistry. The surface markers of myocardial cells were c Tn T. Application of DAPI markers nucleus 1h before transplantation. 3 Rat model of acute myocardial infarction was established by ligation of the anterior descending branch of the coronary artery in SD rats. The electrocardiogram prompt ST segment arched elevation, then appear abnormal Q wave, the surrounding myocardium was pale, and myocardial infarction tissue section were indicating the accomplishment of modeling.4Then rat models were randomly divided into 4 groups with 10 each. The rats with no treatment after AMI was control group.Vein transplantation group and epicardium transplantation group were treated with cardiomyocyte-like cells suspension200ul(cardiomyocyte-like cells 5×106) after AMI via tail vein and epicardium transplantation respectively. The combined transplantion group were transplanted with cardiomyocyte-like cells suspension 200 ul respectively after AMI via tail vein and epicardium. 4 weeks after weight and left ventricular weight in rats, hematoxylin eosin staining to observe SD rats myocardial tissue morphology and distribution of transplanted cells under fluorescence microscope and analysis of the expression of myocardial tissue protein ACE2 by western blot. The measurement data were demonstrated by mean±SD.All data was analyzed by the SPSS17.0. Single factor analysis of variance was used to compare the number of multiple mean. Further two two compared with the LSD test method. A value of P<0.05 was considered as statistical significant.Results 1 Bone marrow mesenchymal stem cells after inoculation 24 h. A large number of suspension cells seen under the microscope, high refractive index, medium turbidity, a small amount of adherent cells, morphological diversity. The cell morphology changed,and finally showed a vortex like growth. The shape is uniform, and the shape is shuttle shape. Passage 48 h, due to trypsin, part of cell shrinkage, off the wall, leading to cell suspension more adherent cells decreased. Then the cell recovery its normal form. 7d, the number of cells reached the maximum. BMSCs morphology was more uniform and had short spindle shape. P3 cells were taken for immunocytochemistry, and the surface markers of bone marrow mesenchymal stem cells CD44+ and CD34- were identified. 2BMSCs was induced by 5-azacytidine to induce cardiac-like cells in vitro. A small number of cells were affected by the toxicity of 5-azacytidine to death. But the proliferation rate was slower than before, and the cells began to merge with each other.After 4 weeks, the cell volume became larger, and showed a long spindle shape. The c Tn T was showed positive expression. 3 Ligation of the anterior descending branch of the coronary artery in rats, the following changes in the heart muscle becomes pale.Electrocardiogram ST segment elevation. Tissue sections showed disorder of myocardial fibers, even rupture, nuclear rupture, fibrous tissue proliferation. 4 The left ventricular mass index in the transplantation group significantly decreased compared with that in the control group. Compared with vein transplantation group and epicardium group, the left ventricular mass index were more significantly decreased in the combined group. 5 DAPI labeled BMSCs transplanted into rats, observed under the fluorescence microscope, the myocardial nuclei were round or oval blue fluorescence. Vein transplantation group,epicardium transplantation group and the combined group were found to have DAPI labeled positive cells in the myocardial infarction area. Their number in the combined group was significantly increased compared with vein transplantation group and epicardium group. 6 H-E staining showed that the myocardial cells were arranged much more in order and the nuclei were more complete in the last three groups comparison with control group, and the damage recovery of combined group was the most significant.7 Compared with control group, the protein expression levels of ACE2 were significantly increased in myocardial tissues in the other groups. Compared with vein transplantation group and epicardium group, the protein expression levels of ACE2 were more significantly increased in the combined group.Conclusions 1 Intravenous transplantation and epicardium transplantation of bone marrow mesenchymal stem cells could promote the infarct tissue repair; 2 The vein transplantation combined epicardial transplantation of bone marrow mesenchymal stem cells promote the infarct tissue repair, inhibit ventricular remodeling more significantly. |
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