| [Objective]By isolating and culturing myocardial cells and building the model of cardiomyoblast cells injury. To investigate whether the wnt/β-catenin signaling pathway is involved in the myocardial cell apoptosis. And bone marrow mesenchymal stem cells were co-culture with injured cardio-myocytes, further to explore wnt/β-catenin signaling pathway and the changes of myocardial cell apoptosis. To provide some experimental results for the bone marrow mesenchymal stem cells transplantation of the damaged myocardial cells.[Methods]The primary cardiocytes in neonatal rat harvested with trypsin digestion and were isolated by different attachment techniques, In order to identify the myocardial cells, the expression of α-smooth muscle actin were tested by immuno-histochemistry assay. ADR (1mg/1) was used to induce the model of cardiomyoblast cells injury. Base on the different damage times the cardiomyocytes were divided into four groups:A: ADR-injured cardiomyocytes for 12h, B:ADR-injured cardiomyocytes for 24h, C:ADR-injured cardiomyocytes for 48h, D:Normal cardiomyoc ytes group, The expression difference of wnt2, β-catenin, p53 in each group were observed by western blot.Bone marrow mesenchymal stem cells were isolated, identified and cultured for further study. Cardiomyocytes were divided into three groups. E:Normal cardiomyocyte group, F:injured cardiomyocyte death group, G:MSCs co-culture with injured cardiomyocyte group. Detection of apoptosis rate in cardiomyocytes by flow cytometer, the expression of β-catenin, c-Myc, p53 protein were tested by western blot.[Results]1. To test the expression of the α-smooth muscle actin in the harvested primary cardiocytes in neonatal rat. The staining results were positive. Showed that the cultivated cells were myocardial cells.2. To detect the protein expression of wnt2, β-catenin, and p53 in group A, B, C, D. The resuluts showed that the expression of wnt2 in group A, B, C were respectively 0.24+0.04,1.15+0.05,0.35+0.05, the expression of P-catenin in group A, B, C were respectively 0.21+0.04, 0.34+0.04,0.71+0.04, the expression of p53 were respectively 0.17+0.03, 0.61+0.40,0.83+0.04. In gourp A, B, C the expression of the three proteins were gradually increasing, all the differences were statistically significant (P<0.01). The expression of wnt2, β-catenin, and p53 couldn’t be detected in group D. The correlation coefficient between Wnt-2 and β-catenin were 0.314, while wnt2 and p53 were 0.584, Significantly positive correlation between p53 and β-catenin expression could be observed (r=0.940), the differences were all statistically significant (P< 0.01). we suggested that wnt/β-catenin signaling pathway plays a signify-cant role in the injured cardiomyocytes process.3. The myocardial cell apoptosis rate of group E were 0.74+0.65, group F were 26.58+3.89, group G were 7.72+0.90. By multiple com-parisons, it showed that the apoptosis rate of group F were higher than group E and G, all the difference were statistically significant (P<0.01).4. The expression of protein β-catenin, c-myc, p53 could not be detected in group E. Compare with group G, the expression of β-catenin, c-myc, p53 protein in the group F were higher(P<0.01), the comparisons of all were statistically significant(P<0.01), The correlation coefficient between β-catenin and c-myc were 0.942, β-catenin and c-myc were 0.852, c-myc and p53 were 0.927. Significantly positive correlation among the expression of β-catenin, c-myc, p53 could be observed, all the differences were statistically significant(P<0.01). It suggested that bone marrow mesenchymal stem cells can inhibit the expression of the apoptosis protein p53 in the damaged cardiomyocytes, and the mechanism may relative to the decreased expression of P-catenin and its downstream protenin c-myc.[Conclusion]The wnt/β-catenin signaling pathway is involved in the myocardial cell apoptosis. Bone marrow mesenchymal stem cells can inhibit the apoptosis of damaged cardiomyocytes, the mechanism may relative to wnt/β-catenin signaling pathway and its downstream protenin c-myc-mediated regulation of p53. |
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