The Relationship Between Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Myocardial Cells And The β-Catenin Of Wnt/β-Catenin Pathway

Objective:Dilated cardiomyopathy is a disease with high mortality rate, and there is no effective treatment measures. In recent years, much research on stem cells treatment technology brings a new dawn for this disease. This study tries to induce the differentiation of bone marrow mesenchymal cells (BMSCs) into myocardial cells, And to see whether the Wnt/beta-catenin signaling pathway is involved in the differentiation Process. In order to provide effective experimental basis for making the best of bone marrow mesenchymal stem cells transplantation to treat this terminal stage dilated cardiomyopathy in our clinical work.Method:Bone marrow mesenchymal stem cells were isolated from bone marrow of SD male rat about200g. BMSCs was cultured by adherent method, identified with flow cytometry (FCM) at the passage3, and calculated the rate of living cells which were counted through trypan blue staining. Choosing a group of BMSCs at the Passage3with high purity to make into cells suspension, then randomly divided into four groups, named group a、group b、group c、group d, make a distinction between each groups with different consistence of5-Azacytidine about0μmol/L,5μmol/L,10μmol/L,20μmol/L. After two weeks, we chose a befitting consistence basing on activity rate of the total cell and the percentage of myocardial cells to induce the differentiation of BMSCs into myocardial cells. The next experiment we divided BMSCs at the passage3into2groups:the experimental group named group A was treated with5-azacytidine of10μmol/L for24hours and cultured with complete medium without5-azacytidine for four weeks. According to the different cultured time, group A were divided into four groups. group A1was the cells of the first weekend, the other groups can deduce the rest from this, group A2was the second one, group A3was the third one, group A4was the fourth one; Meanwhile the control group named group B was also divided into group B1, group B2, group B3, group B4. Those groups from group B without treated with5-azacytidine. In the process of our experimental, we should observe cellular growth and morphological changes, test the expression of myocardial troponin T by immunofluorescence detection. And at the last western blot technology were applied to test the expression levels of β-catenin and cTnT.Result:1. By flow cytometry and immunophenotyPes, BMSCs showed high expression of CD29, CD105which were mesenchymal cell’s relative surface antigen marks, the rate is93.37%and89.08%. Conversely, It displayed weakly expressed CD34, CD45which were hematopoietic cell marks, the rate is0.89%and1.06%. The living rate of BMSCs at the third passage which Cultured by adherent method through trypan blue staining is>95%.2. Passage3BMSCs were treated with different consistence of Oμmol/L 5μmol/L,10μmol/L,20μmol/L5-azacytidine for group a, group b, group c, group d. Two weeks later, we found the living rat of total cells was as fellow; group a was94.2±7.4(%), group b was93.6±7.1(%), group c was93.5±6.7(%), group d was80.1±9.2(%). Through the comparison of eath groups, we found group d was different from other groups. The myocardial cell of all the groups were0(%),0(%),3.33±.58(%),4.33±0.58(%). Results show that5-azacytidine of10μmol/L was The most suitable conditions to induce BMSCs differentiation into cardiomyocytes.3. Immunofluorescence detection was applied to test the specificity cTnT protein of myocardial cells, red cells could be find under the microscope, there was no positive cells in group B and A1. However percentages of the cardiomyocytes cells was3.33±1.53(%) of group A2,19.67±1.53(%) of group A3,25.67±2.1(%) of group A4. Those results were different and significantly. The expression levels of cTnT was measureed by western blot technology in every weekend. The expression quantity of group B were0.824±0.012,0.826±0.138,0.827±0.02,0.827±0.004, as equal as the livel of group A1which was0.819±0.062, other results of group A2, group A3, group A4were1.231±0.017,6.606±0.227,8.511±0.392. Results shows the difference was significant statistically4. The expression of β-catenin was tested by western blot technology. We could found that there is any expression of β-catenin Protein in group B. The expression quantity of group A as follows:A1(1.877±0.044), A2(7.634±0.329), A3(1.084±0.092), A4(0.473±0.025). the difference was statistically significant with eathother.Conclusion:1. Cells which isolated from bone marrow of SD male rat and cultured by adherent method can harvest sufficient quantities of reliable cells, those cells and marrow mesenchymal stem cells were in the same of PhenotyPic expression.2. BMSCs at the third Passage could be induce differentiation into cardiomyocytes after treated with5-azacytidine, and the consistence of10μmol/L was the befitting consistence to induce the differentiation of BMSCs into myocardial cells.3. The expression of cTnT which from the myocardial cell differentied of MSCs formation increased gradually with time, and the myocardial cell also increased.4. During the induction of BMSCs differentiation into myocardial cells, the expression of cTnT and β-catenin exist time difference, we suggesting beta-catenin which was one main member of the Wnt/beta-catenin Pathway may be involved in regulate differentiation of myocardial cells.