The Screen And Verify Experiment Of TSA Promote Mesenchymal Stem Cells Differentiate Into Cardiomyocyte Cells

Background In the applied clinical research of mesenchymal stem cells (MSCs), how to improve the conversion rate of the stem cells differentiate into cardiomyocyte cells was the research hot spots. Studies have shown that, HDAC inhibitors can mediate the differentiation of several cells through inhibited the activity of HDAC. Several studies reported that trichostatin A (TSA) promoted the differentiation of MSCs to the osteoblast and adipocyte cells. In this study, we investigate whether TSA can promote the differentiation of MSCs to the cardiac myocytes.Objective Different concentrations of TSA with different time intervene MSCs were used to screen the suitable method of TSA inhibiteing the HDAC activity of MSCs. Detected the expression of cTnT and Nkx-2.5, GATA-4 gene during the differentiation, verified TSA could promote MSCs differentiated into the cardiac myocytes, which was the basis for the further study on MSCs.Methods and materials1.Determinated the histone deacetylase activity on 6,24,48 hours after the intervenation of 100,300 and 500nmol/L concentrations TSA on MSCs,which was induced by 5-aza before.2.Cultivated the MSCs which after intervened as mentioned before, studyed the morphological changes of MSCs, the expression of cTnT by immunofluorescence and the expression of Nkx-2.5, GATA-4 gene by RT-PCR.Results1.The HDAC activity of MSCs which intervened with TSA had decreased significantly than before (P<0.05); HDAC activity of 300 and 500nmol /L groups were significantly lower than 100nmol/L group (P<0.05),there was no significant difference between 300 and 500nmol/L groups ; the three concentrations of 24 and 48h groups had decreased significantly than 6h group (P<0.05), there was no significant difference between 24 and 48h groups.2.MSCs volume increased after one week intervention, assumed rod-like or spherical, after two weeks the connection were appeared between the inner cell colony and the adjacent cell colony, with the unanimous orientation.The myotube structure was appeared after 3 weeks. However, with the intervention to increase the concentration and time of a marked increase in the number of cell death, cells also lost arrangement consistency.3.The expression of cTnT was significantly increased than MSCs group (P<0.05), and the expression of cTnT of 300 and 500nmol/L group was significantly enhanced than 100nmol/L group (P<0.05).There was no significant difference between the 300 and 500 nmol/L groups.4.The expression of Nkx-2.5 and GATA-4 after the intervenation with TSA was enhanced complied with the increased concentration of TSA(P<0.05), 300 and 500nmol/L group was significantly higher than 100nmol/L group (P<0.05). There was no significant difference between the 300 and 500 nmol/L groupsConclusions1. The HDAC activity of MSCs which intervened with TSA was dose and time -dependent inhibition.2. TSA can promote the differentiation of MSCs into cardiac myocytes.3. 300nmol/L of TSA intervenes MSCs 24 hours is the appropriate method of promoting the MSCs differentiate into myocardial cells in vitro.