不同培养条件对鸡胚胎生殖细胞Oct4启动子DNA甲基化的影响

Effect of Different Culture Conditions on the DNA Methylation Status of Oct4 Promoter in Chick Embryonic Germ Cells

旨在在体外通过胚胎生殖细胞(EGC)和细胞外基质共同构建一个EGC的小生境(niche)模型,通过比较Oct4DNA甲基化的变化,研究niche中特有的表观遗传效应对胚胎生殖细胞自我更新的影响。构建EGC细胞标准培养方案(对照组)和改良培养方案(试验组),采用甲基化特异性PCR(MS-PCR)技术、RT-PCR技术,对比分析两种培养方案中Oct4启动子的甲基化状态,判断基因表达与其CpG岛甲基化的关系,分析DNA甲基化模式与EGC细胞自我更新的关系。结果显示,试验组可扩增出Oct4的非甲基化扩增产物,而对照组为其甲基化扩增产物,试验组Oct4表达水平明显高于对照组。试验组EGC生长状态明显好于对照组。试验表明,在改良培养条件下,Oct4基因启动子DNA甲基化程度较低,且与基因表达水平呈负相关,更有利于维持胚胎生殖细胞的自我更新状态。

It was to establish a niche model for embryonic germ cells（EGCs）through culturing EGCs in extracellular matrix in vitro.By comparing DNA methylation changes in Oct4 gene,we studied the impact of specific epigenetic effects in niche on the self-renewal of EGCs.Chick EGCs were cultured in standard procedure（control group）and improved procedure（experimental group）,respectively.Methylationspecific PCR（MS-PCR）and RT-PCR were performed to compare the methylation status of Oct4 promoter and its expression level in different procedures.The relationship between Oct4 expression and its methylation status was also determined.Moreover,the relationship between DNA methylation patterns of Oct4 and the self-renewal capability of chick EGCs was explored.Results showed non-methylated products of Oct4 could be amplified in experimental group.While in the control group,the products of PCR amplification were methylated.The level of Oct4 expression was significantly higher in experimental group than in control group.Similarly,the growth state of EGC was significantly better in the experimental group than in the control group.In the improving culture conditions,the methylation level of Oct4 promoter is much lower,and there was a negative correlation between the level of Oct4 expression and its methylation status.These results indicated that low methylation and high expression of Oct4 can promote the self-renewal maintenance of EGCs.