| Objectives Rabbit bone marrow mesenchymal stem cells were used as seed cells to induce into cardiomyocyte-like cells and endothelial cells respectively to construct vascularized tissue engineering myocardial tissue in vitro.To provide theoretical basis and technical support for clinical treatment of myocardial infarction through tissue engineering and cell transplantation.Methods 1 The bilateral femur and tibia of ordinary Japanese white rabbits were isolated aseptically.Rabbit bone marrow mesenchymal stem cells were isolated by Percoll and identified the surface markers CD90,CD105 and CD45 by flow cytometry.Rabbit bone marrow mesenchymal stem cells were induced into endothelial cells with endothelial cell conditioned medium(ECM),and the surface markers CD34 and CD31 were identified by immunocytochemical staining.The morphological changes of rabbit bone marrow mesenchymal stem cells and endothelial cells were observed under microscope.2 Rabbit bone marrow mesenchymal stem cells were induced into cardiomyocyte-like cells with 5-azacytidine.The surface marker c TNT was identified by immunocytochemical staining,and the morphological changes of the cells were observed under microscope.3 Cardiomyocyte-like cells and endothelial cells were labeled with DAPI fluorescent dye and CM-DIL fluorescent dye respectively.4 The cardiomyocyte-like cells and endothelial cells were co-cultured in different proportions(1?0,0?1,1?1,1?2).5 The cells of different groups were inoculated on matrigel matrix glue and collagen sponge to observe the vascularization ability of cell-scaffold composite.6 SPSS 23.0 statistical software was used to analyze the experimental data.The measurement data were expressed in the form of mean±standard deviation.The comparison of the means of multiple samples was performed by single factor ANOVA,with the significant difference in P<0.05.Graph Pad Prism 8.0 software was used to draw the graphics.Results 1 Flow cytometry showed that rabbit bone marrow mesenchymal stem cells could be obtained by using percoll separation solution.The surface markers of CD90 and CD105 were positive,while CD45 was negative.Under microscope,the morphology of rabbit bone marrow mesenchymal stem cells was bright,round and refractive for 48 hours.The first generation of rabbit bone marrow mesenchymal stem cells were of different length and short fusiform.Contains a small amount of impurity cells,the second generation of rabbit bone marrow mesenchymal stem cells,the cells were fusiform,the third generation of rabbit bone marrow mesenchymal stem cells,the volume of cells was larger than the original cells,and the shape becames long fusiform and polygonal.2 Endothelial cells could be obtained from rabbit bone marrow mesenchymal stem cells induced by endothelial cell conditioned medium(ECM),and identified by immunocytochemical staining.The results showed that some endothelial cells began to adhere 48 hours later,the morphology and size of endothelial cells were different and showed cord-like changes.The results showed that the induced cells expressed CD31 and CD34,microscope.In the first generation,the morphology of the cells became short and thin,with a few bright spots;when the cells were passed for the second passage,the number of cells increased significantly and arranged as "paving stone";in the third generation,the cells showed long fusiform changes.3 When cardiomyocyte-like cells were labeled with DAPI fluorescent dye,the nuclei were marked blue,the endothelial cells were labeled with CM-DIL fluorescent dye,and the cell membrane was marked red.4 Rabbit bone marrow mesenchymal stem cells were induced into cardiomyocyte-like cells with 5-azacytidine of 10umol/l.Immunocytochemical staining was used to identify the expression of myocardial marker c TNT.The cells induced by 5-azacytidine for 24 hours showed polygonal shape,uneven distribution and slow growth.After three days of induction,the volume of cells was larger than before,and after five days of induction,the cells began to protrude pseudopodia of different length.After seven days,the cells showed obvious pseudopodia,occasionally connected with each other,and the cell body showed a state of highlight.After three weeks of induction,the number of cells aggregated gradually decreased.After four weeks,obvious changes in cell morphology and a significant decrease in the number of cells were observed.5 The cell-scaffold material complex was constructed in vitro,and different groups of cells were inoculated onto Matrigel matrix glue.It was observed that the vascular reticular formation ability of cardiomyoid cells and rabbit bone marrow-derived endothelial cells was the strongest when co-cultured at 1?2,while on the collagen sponge scaffold,only the cells grew on the collagen sponge and no vascular reticular structure was observed.Conclusions 1 Endothelial cells can be obtained from rabbit bone marrow mesenchymal stem cells induced by endothelial cell conditioned medium.2 Cardiomyocyte-like cells can be obtained from rabbit bone marrow mesenchymal stem cells induced by 5-azacytidine.3 The vascular cell-scaffold material complex is constructed in vitro,and matrigel matrix glue is more suitable than collagen sponge in the selection of scaffold materials.4 In terms of the ability of cell-scaffold material complex to form vascularization,the vascular reticular structure is the strongest when cardiomyocyte-like cells and endothelial cells are co-cultured at the ratio of 1?2.Figure 15;Table 2;Reference 118... |
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