HDAC Inhibitor Trichostatin A Promotes The Specialized Differentiation Of Mesenchymal Stem Cells And Investigate To Mechanism Of Acetylated Action

BackgroundSubstitution therapy by cells is an effective method to treat heart attack. Marrow mesenchymal tissue cells (MSCs) are the first choice to substitute the damaged cardiac muscle cells, because MSCs are easily obtained, multiplied, almost short of anti-host response and do not arouse bioethical limitations. Up to now, there are mainly two approaches to induct MSCs differentiation to cardiac myoid cells: one is to lead the induction by chemicals such as 5-azacytindine or amphotericin (AMPH); and the other is to simulate a microenvironment of cardiac muscle cells in vitro. According to reported materials and the early research of our project team, in the process of MSCs specification to cardiac muscle cells, we find that there is an interacted regulation network organized by heredity and superficial genetic modification, in which remodeling of chromatin, caused by acetylation balance, plays a vital part. The early experiments of the team have broken the balance between histone acetylation and acetylation modification by blocking the expression of GCN5 in MSCs. The decrease of the genes—GATA4 and NKx2.5—in specified cardiac muscle demonstrates that the differentiation from stem cells to cardiac muscle cells has been blocked, which is a proof that acetylation modification of histone plays a vital part in process of differentiation to cardiac myoid cells. Through the intervention of TSA (the inhibitor of de-acetylase) to MSCs that corresponds with the results of the experiments to block the expression of Gcn5 in MSCs, it is identified that the"open and close"mechanism regulates genetic expressions in the process of MSCs being specified to be cardiac muscle cells.ObjectiveThe project is to specify MSCs to be cardiac myoid cells, to identify the regulation mechanism of acetylation in the process of MSCs specification, and to depict genetic network information related to Gcn5 in the process of specifying MSCs through idio-precipitation of Gcn5 proteinum by use of chromatin co-immunoprecipitaion.Materials and Methods1. Take femoral bone marrow and shin bone marrow of Wistar mouses. Dissociate, cultivate and multiply vitro MSCs by adherence method; take the hearts of the newly born Wistar mouses (1-3 days old). Dissociate, depurate and cultivate cardiac muscle cells by concoction dissociation method, speed differential adherence method and reagent chemical inhibition method.2. Divide the experiment into three groups: original control group (MSCs), induction group and TSA inhibition group. Detect the early transcription factors of cardiac muscle cells—GATA-4, NKx2.5, and MEF2c by the method of real time fluorescence PCR based on the method of SYBR.GREENI, and observe changes of specified Troponin T in cardiac muscle by the method of immunofluorescence and Western-blot.3. Induct MSCs by 5-azacytidine to encourage the differentiation of cardiac muscle cells in MSCs; establish co-culture system of MSCs and cardiac muscle cells in the transwell snap-on culture dish in order to simulate the microenvironment of similar cardiac muscles transplanted by MSCs; and conduct intervention by the inhibitor TSA of de-acetylase of different concentration.4. By co-immunoprecipitation of chromatin, precipitate related genes of cardiac muscle cells bound with Gcn5 proteinum. Design primed DNA by targeting the idio-genes of cardiac muscle cells—GATA-4, NKx2.5, and MEF2c, and observe the expression of idio-genes of cardiac muscles which are related to Gcn5 in the process of MSCs specification. Results1. One week after the co-culture of MSCs and vibrating cardiac muscle cells, MSCs begin to differentiate to sample cardiac muscle cells, which indicates that a microenvironment of cardiac muscle has been established.2. Co-culture group: One week after the co-culture of MSCs and vibrating cardiac muscle cells, MSCs begin to differentiate to sample cardiac muscle cells. Real time fluorescent quantitation PCR indicates that, after co-culture and inhibition by TSA of different concentration, disparity 500nmol/L TSA is significant (P<0.05) in the comparison among the groups of expression levels of early transcription factors of cardiac muscle and the expression in a certain range of concentration shows a rising tendency with the increase of TSA concentration; by immunofluorescent coloration and Western-blot detection, the expression of specified troponin T,Cx43 and MHC of cardiac muscle is visible in amicula. The disparity is significant (P<0.05) in the comparison between the differentiation rate of amicula and the expression quantity of troponin T when the concentration of TSA inhibition groups changes.3. 5-aza induction group: when MSCs are inducted by 5-aza and inhibited by TSA of different concentration, the disparity is significant (P<0.05) in the comparison between the expression level of early transcription factors of cardiac muscle and the specified troponin T of cardiac muscle. The expression is in positive correlation to TSA concentration in a certain range of concentration.4. By use of chromatin co-immunoprecipitation, related genes of cardiac muscle cells bound with Gcn5 proteinum are precipitated. The expression of idio-genes of cardiac muscle related to Gcn5—GATA-4, NKx2.5 are observed in the process of MSCs specification. ConclusionHDAC enzyme inhibitor promotes the process of MSCs specification to cardiac muscle cells. Acetylase activity is in positive correlation to the rate of cell specification in a certain range of TSA concentration. There are expressions of idio-genes of cardiac muscle related to Gcn5—GATA-4, NKx2.5 in the process of MSCs specification. A regulation network exists in the interaction between proteinum and factors.