BIX-01294对山羊胎儿成纤维细胞生长状态和组蛋白H3K9二甲基化的影响

Effect of BIX-01294 on Growing Status and H3K9 Dimethylation of Goat Embryonic Fibroblasts

为探讨BIX-01294对供核细胞的影响,使用不同浓度的BIX-01294处理山羊胎儿成纤维细胞(Goat embryonic fibroblasts,GEFs),并检测其细胞活力、细胞周期、细胞凋亡、H3K9me2水平和多能性基因的mRNA水平。细胞活力检测结果显示,BIX-01294浓度在1.0μmol/L以下不会影响细胞活力,2.0μmol/L时24h和48h处理组的细胞活力显著下降(P<0.05),≥4.0μmol/L时所有处理组的细胞活力均极显著下降(P<0.01)。细胞周期和凋亡检测结果显示,H3K9me2下调会引起细胞周期的停滞和正常细胞的减少,其中BIX-01294在大于2.0μmol/L时G0/G1期细胞的比例极显著升高(P<0.01),4.0μmol/L时凋亡细胞极显著增加(P<0.01)。处理后的细胞H3K9me2水平均极显著下降(P<0.01),但大于1.0μmol/L的处理浓度能获得较好的下调效果。处理后细胞Oct4、Sox2和Nanog的mRNA水平均有不同程度的增加,其中Oct4和Nanog的mRNA水平增加极显著(P<0.01)。说明:采用1.0μmol/L BIX-01294处理山羊胎儿成纤维细胞,可以下调H3K9me2水平,增加G1/G0期细胞比例,上调细胞多能性基因表达量,而且不影响细胞活力。

To study the effect of BIX-01294 on donor cells, we conducted a series of tests on goat embryonic {ibroblasts （GEFs）, including cell viability assays, cell cycle and cell apoptosis test, immunostaining of H3K9me2 and mRNA expression of pluripotent genes. These tests lay the foundation for future research of the effect of BIX 01294 on development of cloned embryos by using BIX-01294-treated donor cells. Cell viability assays showed that there was no significant decrease of cell viability when less than 1.0 -mol/L BIX-01294 was used. But cell viability of both 2.0 μmol/L group （at 24 and 48 h） and 4.0 μmol/L group （at 24,48 and 72 h） decreased significantly （P〈0.05 and P〈0.01, respectively）. Down-regulating H3K9me2 caused cell cycle arrest with significantly increased G0/G1 cells （P〈0.01） at 2.0 and 4.0 μmol/L, and decreased normal cells with significant difference at 4.0μmol/L （P〈0.01）. The level of H3K9me2 decreased significantly P〈0.01） in all treated groups.However, the concentration of BIX-01294 at more than 1. 0μmol/L presented a better inhibiting effect. We also found significant increase of mRNA expression of Oct4 and Nanog （P〈0.01）. Based on these results, BIX-01294 at 1.0μmol/L was preferred because all effects of down-regulating H3Kgme2, increasing G0/G1 cells and mRNA expression of pluripotent genes and no decrease on cell viability were detected at same time.