Effect Of Nabu On Cell Cycle, Vitality And Apoptosis And Histone Acetylation Of Bovine Fibroblast Cells

Somatic cell nuclear transfer technique is a kind of animal embryo engineering and this technology is becoming more important in massive production of in vitro embryos, however, this technology has still relatively low efficiency and cannot be used in producing cloneing animal.One important reason is the incomplete reprogramming of donor cells. So this experiment was designed to study donor cells histone acetylation for reprogramming. In view of the above problem, the bovine fibroblasts passaged 6 generations were collected as the donor cell and the oocytes as the acceptor,geting these result and conclusion:1.Bovine somatic fibroblasts passaged 6 times were seeded into DMEM plus 10%FBS containing 3mmol/L of NaBu for 0,48,72,96h. Cells were subsequently subjected to test for histone acetylation of H3K9,14 levels by confocal microscopy.To investigate the effect of NaBu treatment donor cells on in vitro development of bovine nuclear transfer(NT)embryos which constructed from bovine fibroblast cells.The results showed that NaBu increased the levels of histone acetylation of H3K9,14 after bovine fibroblast Cells treated with 3mmol/L NaBu with the increase of treatment time.3mmol/L NaBu treatment of donor cells for 48h markedly increased cloned embyo development potential,compared to controls(32.5%vs22%,p<0.05).Conclusion:3 mmol/L NaBu 48h treatment donor cell are more likely to be oocytes reprogramming, remarkably raised the cloned embryos in vitro development ability, that may be through elevate the donor cell histone acetylation level to promote the donor cell reprogramming.2.We treated donor cells with histone deacetylase inhibitor NaBu, detection the histone acetylation of cells and the influence of characteristics.Try to determine the optimal concentration and treatment time of NaBu.we will get the result,which donot affects somatic cell biology characteristic and enhance the donor cell histone acetylation. thereby providing a more efficiency method for somatic cell cloning. In this experiment, different time of trentment of NaBu was used to treat bovine fibroblasts for 0,48,72,96 h, by flow cytometry, and agarose electrophoresis respectively, to test the effect of NaBu on cell cycle,cell shape and apoptosis at the same time, using the MTT for detecting cell vitality after treatment with NaBu. The results were as follows:The rate of G0/G1 phase cells treated with NaBu(3mmol/L0-96h) had slightly lowed compared with the untreated group;From the agarose gel electrophoresis patterns we found that cells treated by NaBu 24,48h had no "ladder" phenomenon, While the phenomenon began from group of 72h. The proportion of apoptosis treated by 72h and 96h groups significantly increased compared with the control group.At the same time,we treated the cells with Annexinâ…¤and PI,with the increse of the treatment time,the proportion of apoptosis significantly increased compared with the control group. Anyhow, the experiment result showed that with NaBu 3mmol/treated donor cell 48h, the cell apoptosis was relatively low,more than 75% cell in Go/G1phase, but longer treatment time resulted in DNA fiagmentation and significantly increased cell apoptosis, could not be used for the production of SCNT in this experiment.