VPA对牛胎儿成纤维细胞体外培养的影响

Effect of valproic acid( VPA) on the in vitro culture of bovine fetal fibroblasts

为了研究组蛋白去乙酰化酶抑制剂(HDACIs)丙戊酸(VPA)处理对牛胎儿成纤维细胞体外培养的影响,试验采用培养到第3代的牛胎儿成纤维细胞经VPA处理后,进行细胞生长曲线绘制及形态、活力、细胞周期的检测,并通过Real-time PCR检测Oct4和Cdx2的表达情况,经梯度浓度的VPA处理牛胎儿成纤维细胞,于24 h后观察细胞形态。结果表明:当VPA浓度高于0.5 mmol/L时,细胞开始出现异常形态,死细胞数目增多。0.5 mmol/L VPA处理牛成纤维细胞24 h可以显著抑制细胞活力,可将60%以上细胞的周期阻滞在G0/G1期;但处理48 h将导致细胞染色体整倍率显著下降。与对照组相比,VPA处理后,Oct4的表达量显著升高,而Cdx2基因的表达量则显著降低。说明用HDACIs VPA处理过的牛成纤维细胞作为体细胞核移植的供体细胞,可能更有利于克隆胚胎重编程及发育。

To study the effect of valproic acid（ VPA） as a kind of histone deacetylase inhibitors（ HDACIs） on the in vitro culture of bovine fetal fibroblasts. The bovine fetal fibroblasts cultured to the third generation,were treated with different concentrations of VPA for drawing the cell growth curve and the detections of cell morphology,cell vitality and cell cycle,and the expression levels of the Oct4 and Cdx2 genes were detected using real- time PCR. The bovine fetal fibroblasts were treated with the gradient concentrations of VPA for the observation of cell morphology after 24 h. The results showed that the bovine fibroblasts began to appear abnormal morphology and the number of dead cells increased when the concentration of VPA was higher than 0. 5 mmol / L. The viability of bovine fibroblasts was significantly inhibited after the treatment with 0. 5 mmol / L VPA for 24 h,and the cell cycle of more than 60% fibroblasts cell cycle could be blocked in G0 / G1 phase; but the chromosome ploidy of the fibroblasts was significantly with the treatment for 48 h. Compared with the control group,the expression of Oct4 was significantly increased after VPA treatment,while the expression level of Cdx2 gene was significantly decreased. The results indicate that the bovine fibroblast cells treated by HDACIs as donor cells for somatic cell nuclear transplantation,may be more conducive to the epigenetic reprogramming and the development of the cloned embryos.