RG108对猪胎儿成纤维细胞甲基化水平及克隆胚胎发育的影响

Treatment of Porcine Fetal Fibroblast with RG108 on Genomic DNA Methylation Levels of Cells and Development of Cloned Embryos

本研究使用RG108处理猪胎儿成纤维细胞(Porcine fetal fibroblast,PFF),研究其对细胞甲基化水平和核移植效率的影响。使用0.05、0.5、5和50μmol·L-1RG108分别处理细胞24、48和72 h后,用高效液相色谱和亚硫酸氢盐测序(Bisulfite sequencing PCR,BSP)法分析细胞整体甲基化水平和印迹基因H19和IGF2R的DMR区甲基化率,并检测RG108处理对细胞生长、凋亡、染色体变化及随后的核移植效率的影响。整体甲基化水平结果通过两因素方差分析,发现RG108处理浓度与处理时间无交互作用,处理浓度间细胞甲基化水平差异不显著,但处理时间却能显著的影响甲基化水平,且50μmol·L-1RG108处理细胞72 h后整体甲基化水平显著低于对照组。BSP分析结果表明,5和50μmol·L-1处理PFF 72 h后H19的DMR区域甲基化率显著降低。RG108对细胞生长有一定的抑制作用,且0.5、5和50μmol·L-1组经处理72 h后凋亡比例显著提高,但对细胞的染色体数目没有显著影响。PFF经5和50μmol·L-1RG108处理72 h后能显著提高卵裂率和囊胚细胞数,且5μmol·L-1组囊胚率也显著提高。结果表明,RG108降低细胞整体甲基化水平呈时间依赖性,且同时降低H19的DMR区域甲基化率。PFF经5μmol·L-1浓度处理72 h组能显著提高核移植胚胎效率。

This experiment was conducted to study the effect of treatment of PFF as donor cells with RG108 on their methylation levels and nuclear transplantation efficiency. Four concentrations, 0.05, 0.5, 5 and 50 μmol· L^-1 of RG108 were selected. PFF was treated for 24, 48,72 h respectively, and the cellular methylation levels were determined by HPLC and BSP methods. The situation of cell growth and apoptosis was observed by using the MTT method and fluorescent microscope observation method. The cellular chromosome and nuclear transfer efficiency after treatment with four concentrations of RG108 for 72 h were detected using Giemsa staining approach. The results of the overall methylation levels were tested with two-way ANOVA analysis, which showed that the interaction between concentration and the incubation time of RG108 was not significant. The incubation concentration had no significant effect on the methylation level, but the treatment time did. The PFF treated with 50 μmol· L^-1 RG108 for 72 h, its methylation level was lower than control group significantly. The BSP analysis results showed that the H19 DMR region methylation was significantly lower after the treatment of 5 and 50μmol· L^-1 for 72 h. After RG108 treatment for 72 h, the cell apoptosis rates of 0.5,5,50 μmol· L^-1 group increased significantly, but the chromosome numbers of cells were not significantly affected, showing that RG108 inhibited cell growth. After the PFF cells treated with 5 and 50 μmol· L^-1 RG108 for 72 h, the cell numbers of blastocyst and cleavage rate increased significantly, and for 5 μmol· L^-1RG108 treatment, it could significantly enhance the blastocyst rate. The results indicated that PFF incubated with RG108 could result in a reduction of the cellular overall in the H19 DMR region as well of nuclear transfer. methylation levels in a time-dependent pattern PFF treated with 5μmol· L^-1 RG108 for 72 h and a reduction in the methylation levels could significantly improve the efficiency