| Objective:To introduce a new method to induce mice mesenchymal stem cells to differentiate into insulin-secreting cells and explore the appropriate concentration of the new revulsant (TSA).Methods:The C57BL/6 mice mesenchymal stem cell line was cultured in vitro and divided into six groups before treated with different concentration TSA (group A:PBS; group B:DMSO; group C:25 nM; group D:50 nM; group E:100 nM; group F:200 nM). After exposured to the different cultured medium for ten days during two stages,the cells were detected by follow methods:1. The insulin-secreting cells in each group were identified by Dithizone Staining and the results of TSA-treated groups were calculated with immunohistochemical half quantitative analysis.2. The insulin secreted by insulin-secreting cells in each group was identified by Immunofluorescence and the mean fluorescence intensity of insulin in TSA-treated groups was compared.3. The contents of insulin in each group were quantified by Enzyme Linked Immunosorbent Assay.4. The appropriate concentration of TSA was determined according to the results of above three points.Results:1. TSA promoted bone marrow mesenchymal stem cells in mice to differentiate into insulin-secreting cells which producted insulin in 10 days.2. Image analysis of insulin-secreting cells immunohistochemistry and immunofluorescence showed that the insulin staining positive area, area ratio,total density of insulin expression and mean fluorescence intensity of insulin in group C were significantly higher than those in the other TSA-treated groups (allP<0.05)3. With the concentrations of TSA gradually increased, the contents of insulin reduced accordingly. The contents of insulin in group C were significantly higher than those in the other TSA-treated groups (all P<0.05)Conclusion:It was confirmed that TSA promoted bone marrow mesenchymal stem cells in C57BL/6 mice to differentiate into insulin-secreting cells in 10 days and the appropriate concentration of TSA was 25nM.
|
PDF Full Text Request