Apoptosis Of Bovine Fetal Fibroblast Cells Induced By Trichostatin A

Production of healthy and viable cloned animals by somatic cell nuclear transfer is currently a hot field in life science research. At present, such problems as low cloning efficiency and dysplasia are major problems in somatic cell cloning. These problems are closely related with the incomplete epigenetic reprogramming of the donor cells by oocyte cytoplasm. In order to improve the efficiency of cloning and the development of cloned embryos, many researchers have tried to treat donor cells with varieties of reagents that can change the modification of DNA methylation or histone modification. However, toxical effect of of these reagents on embryonic development remains controversial.In this study, trichostatin A (TSA), a histone acetylation enzyme inhibitor, were used to study its effect on donor cells and its mechanism in inducing donor cell apoptosis. Different concentrations of TSA (0μM,0.05μM,0.10μM,0.20μM,0.40μM,0.80μM,1.60μM,3.20μM) were used to treat bovine fetal fibroblasts for24h,48h and72h. Then, cell morphology was observed with a inverted phase contrast microscope, cell proliferation activity was detected by MTT colorimetry, cell cycle was measued by using flow cytometry after PI staining, and cell apoptosis was detected with flow cytometry after AnexinV/PI double staining. The transcription levels of apoptosis related genes, including bcl-2, bax, cytc, caspase-9and caspase3, were analyzed by Realtime PCR. The DNA methylation status at apoptosis related gene promoter regions were detected by bisulfite sequencing.The main results were as follows:1. The effects of TSA treatment on cell morphology, proliferation, cell cycle and apoptosisTreatment of bovine fetal fibroblasts with TSA lower than0.4μM did not affect their shape, but TSA higher than0.4μM made the cell body retract, and cytoplasm turn dark. With the increase of TSA concentration, more cells detached from culture dish.Proliferation activity of bovine fetal fibroblast cells were inhibited by treating with TSA for24h,48h and72h, and the inhibition has dose-dependent and time-dependent effects. After treatment for24h,48h and72h, cell relative proliferation rate decreased from98.64%to60.1756%, from98.69%to39.20%, and from96.2061%to22.4723%, respectively. The percentage of cells in G0/G1phase significantly increased, while the percentage of cells in S and G2/M phase significantly reduced after TSA treatment for24h,48h and72h. When the TSA concentration was lower than0.8μM, the percentage of cells in G0/G1phase gradually increased with increasing concentrations of TSA, while cells in S and G2/M phase decreased with increasing concentrations of TSA. When TSA concentration was higher than0.8μM, the percentage of cells in G0/G1phase slightly decreased and the percentage of cells in S and G2/M phase increased. Therefore, bovine fetal fibroblasts can be arrested in G0/G1phase effectively with0.8μM TSA.TSA treatment induced bovine fetal fibroblasts apoptosis. After24h, early apoptosis rate increased from4.55%to21.20%, late apoptosis rate increased from1.31%to6.08%, cell death rate increased from0.44%to2.93%, and living cells gradually decreased from93.7%to69.09%. treatment for48h and72h further increased the early and late apoptosis rate, and further reduced living cells rate.2. The effects of TSA treatment onapoptosis related gene expression and the DNA methylation status at apoptosis related gene promoter regionsAfter0.80μM TSA treatment for24h,48h, and72h, the expression of apoptosis inhibiting gene Bcl-2decreased with time extending, and the DNA methylation level at its promoter region increased after48h but showed no obvious changes after24h and72h. The expression of apoptosis promoting gene bax increased with time extending, and the DNA methylation level at its promoter region increased after48h treatment but showed no obvious changes after24h and72h treatment. The expression of apoptosis promoting gene cytc, Caspase-9and Caspase-3increased with time extending.In conclusion, this study demonstrates that TSA can induce apoptosis of donor cells by changing the epigenetic modification of apoptosis related gene promoter regions.