| Since Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal in1997, and the technology of mammalian somatic cell nuclear transfer (SCNT) has entered a new stage. Although SCNT created a broad application prospects for basic research, there were still many problems need to be solved, such as low efficiency of cloning and high likehood of abnormal development of cloned animals. It was shown that abnormal epigenetic modifications such as histone modifications were revealed in cloned embryos or offspring, which suggests that epigenetic modifications incomplete or abnormal of donor nucleus might give rise to abnormal development of cloned embryos. Many HDACis have been applied to improve somatic cell nucleus reprogramming, however there were great differences among them and the majority of results were in mice. Although HDACi could improve reprogramming of somatic cell nucleus, they could have toxicity to donor cells. Thus, it is probably important for improving cloning efficiency and for modifying aberrant genomic reprogramming to discover a type of HDACi which has no/or low toxicity.In this study, the effect of two kinds of HDACi, TSA and VPA, on culture of bovine fibroblast cell in vitro was estimated. Our aim is to make bovine fibroblast more suitable to act as nuclear donor cells through interfering histone modifications caused by HDACi. This research will benefit for improving the efficiency of assisted reproductive technologies and cloning.In this paper, the third generation of cultured bovine fibroblasts for the study, such as cell growth curve, morphology, vitality, cell cycle, and the expression of Oct4and Cdx2. The results showed that:The bovine fibroblast cells were treated with different concentrations of TSA or VPA, then cell growth curve were examined, the results show that the effect of HDACi was dose dependent. The bovine fibroblast grew more rapidly after48h treatment and reached peak after96h. The morphology of bovine fibroblast treated by different concentrations of TSA and VPA was observed.The bovine fibroblast began to appear abnormal morphology and increase the number of dead cells when the concentration of TSA was higher than50nM or the concentration of VPA was higher than0.5mM. The bovine fibroblast grew slowly after48h treated by50nM TSA or0.5mM50nM VPA, but the outline of cells were still clear. However, with the increasing of the concentration, the phenomenon of cell aging was obvious and the number of dead cells was increased. The result of MTT showed that the viability of bovine fibroblast was inhibited significantly after treatment by50nM TSA or0.5mM VPA for24h.Bovine fibroblast chromosomal composition was affected by HDACi. The diploid composition of the cells decreased significantly treated by50nM TSA or0.5mM VPA for24h. The VPA group was lower than TSA group but the difference was not significant. The cell cycle was detected by flow cytometry showed that up to60%of cells were arrested in G0/G1phase treated by50nM TSA or0.5mM VPA for24h. Real-Time PCR results show that compared with the control group, after TSA or VPA treatment, the expression of Oct4increased significantly, while the expression level of Cdx2gene decreased, but TSA and VPA treatment group had no significant difference.The results suggested that HDACi treated bovine fibroblast cells may benefit the epigenetic reprogramming and the cloned embryos development when it was used as donor cells in somatic cell nuclear transfer. |
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