TSA对滩羊成纤维细胞作为供体细胞的作用

Effect of TSA on Fibroblast Cell as Donor Cell in the Tan Sheep

为探索成纤维细胞作为供体细胞的潜能和TrichostatinA(TSA)处理供体细胞的适合浓度,提高克隆胚胎的发育水平。本研究分别利用100、50、25、10ng·mL-1TSA处理滩羊成纤维细胞,观察细胞形态,统计细胞活率,利用流式分选技术分析处理前后细胞的周期时相,同时通过间接免疫荧光法检测细胞乙酰化水平,并以经过10～50ng·mL-1TSA处理的成纤维细胞作为供体细胞,检测其重构胚发育水平。结果发现,当TSA的浓度达100ng·mL-1时,细胞大量死亡,不同浓度TSA处理细胞24h后,细胞周期被明显抑制在G0/G1期,乙酰化水平明显高于对照组,其中供体细胞经10ng·mL-1TSA处理的克隆胚胎卵裂率和囊胚率明显升高(P<0.05,(85.2±3.4)%vs(68.6±6.7)%,(35.6±5.7)%vs(10.4±8.3)%)。结果表明,经10ng·mL-1TSA处理的滩羊成纤维细胞周期同步化明显,乙酰化维持较高水平,重构胚胎发育水平明显提高,适合作为成纤维细胞的处理方法和浓度。

In order to investigate the potential of fibroblasts as donor cells,and find out the optimal concentration of TSA used to treat fibroblasts,and further to improve the developmental level of cloned embryos,the fibroblasts were treated with 100,50,25,10 ng·mL-1 TSA,cell morphology and viability were investigated.Flow cytometry were adopted to analyse cell cycle.Acetylation level was detected by indirect immunofluorescence,and the developmental level of cloned embryos was evaluated.Most cells treated with 100 ng·mL-1 TSA were dead,but a significant increase of acetylation level was detected;the cell cycle was arrested at G0/G1 checkpoint with the existence of TSA;generally,there was a higher proportion of cleavages and blastulas（P0.05,（85.2±3.4）% vs（68.6±6.7）%,（35.6±5.7）% vs（10.4±8.3）%）when the cells were treated with 10 ng·mL-1 TSA.The results suggested that acetylation level,cell morphology and embryonic developmental level were improved and optimal for the donor cells treated with 10 ng·mL-1 TSA.