Research On Histone Acetylation Mechanisams Of Regulating The Process Of MSCs Specially Differentiating Into Cardiomyocytes

Objective:The study is to screen valid plasmid targeted to Gcn5 among the constructed recombinant plasmids; and to detect weather it could silence Gcn5 gene expression. To confirm the effective plasmid which could cause low-grade acetylation. Then to explore the disbalance of histone acetylizad modification result in the effective plasmid could regulate MSCs differentiation in vivo and in vitro. Finally establish a technique platform for the switch which regulates the gene expression in the process of MSCs differentiation.Methods and materials:1. Screen an effective plasmid, transfect different plasmids into MSCs, and then investigate the cell morphologic change, HAT activity and the expression of Gcn5.2. Confirm weather the plasmid ZJ3 could cut down the level of histone acetylation, transfect the MSCs with shRNA-ZJ3 and then detect the HAT activity of MSCs.3. Analysis weather the low-grade acetylation of histone could regulate the MSCs differentiation, transfect the MSCs induced by 5-aza or not with shRNA-ZJ3 and then detect the cell morphologic change, HAT activity, the expression of Gcn5 and GATA-4 cross-linked to H3 peptide chain, and the ultra microstructure of MSCs.4. Transfect the MSCs induced by 5-aza with shRNA-ZJ3, and transplant MSCs into myocardium which marked with GFP or DAPI. Two weeks later, collect the myocardium transplanted by MSCs, and detect the HAT activity, expression of gene Gcn5 and GATA-4 and the expression of protein Cx43, MHC, and Gcn5.Results:1. In MSCs transfected by different plasmids, some of MSCs which was transfected by shRNA-ZJ3 has a significant change in cell morphologic, low-grade activity of HAT and low-expression of protein Gcn5.2. HAT activity of MSCs transfected by shRNA-ZJ3 is lower than normal MSCs.3. MSCs induced by 5-aza grow vigorously, but the MSCs transfected by shRNA-ZJ3 grow flaggingly. HAT activity of MSCs induced by 5-aza shows at high-grade; however in the MSCs transfected by shRNA-ZJ3, HAT activity shows at a low-grade. The expression of Gcn5 and GATA-4 cross-linked to H3 peptide chain is obvious in the MSCs induced by 5-aza, and it is weak in the MSCs transfected by shRNA-ZJ3. Myofilament was detected in the cytoplasm of MSCs induced by 5-aza; and some apoptosis cells were found in the sample of MSCs transfected by shRNA-ZJ3; in the transfected MSCs, the percentage of heterochromatin is larger than in the normal MSCs and induced MSCs.4. In the myocardium transplanted by MSCs which transfected by shRNA-ZJ3, HAT activity drop off obviously; the expression of gene Gcn5 and GATA-4 depressed significantly; and the expression of protein MHC, Cx43 and Gcn5 expressed also at a low level.Conclusion:1. shRNA-ZJ3 is the effective plasmid targeted to Gcn5.2. Gcn5 blocked by shRNA-ZJ3 can obstruct the modification of histone, and break the balance of histone acetylation.3. The balance of histone acetylation plays an important role to regulate the process of MSCs differention into cardiomyocytes.4. In the myocardial microenvironment, the differentiation activity and priming of MSCs is closely related to the HAT activity.5. In the myocardial microenvironment, silenced MSCs resulted from the disbalance of acetylation could not differentiate into cardiomyocytes.6. The study established a technique platform for the research on histone acetylation mechanisams of regulating the gene expression in the process of MSCs differentiation successfully.