胰岛素样生长因子1拮抗高糖对植入前期鼠胚印迹基因H19/Igf-2的影响

背景：课题前期研究已证实100 μg/L胰岛素样生长因子1能拮抗体外30 mmol/L高糖毒性引起的凋亡,可改善高糖环境对孕母/胎儿的危害。但这一改善胚胎早期发育的营养环境的发生机制和分子机制却不甚清楚。目的：结合前期工作基础,从表观遗传学角度入手,观察胰岛素样生长因子1拮抗早期高糖环境对小鼠胚胎印迹基因H19/Igf-2甲基化水平和表达的影响。方法：通过建立孕娠糖尿病小鼠模型,取小鼠2细胞胚胎等分为实验组和对照组;对照组置于含30 mmol/L葡萄糖的KSOM培养基内进行体外高糖逆境培养,实验组额外添加100 μg/L胰岛素样生长因子1处理。体外发育至囊胚期,检测胚胎印迹基因H19,Igf-2的表达和印迹控制区DNA甲基化水平。结果与结论：实时定量PCR分析表明,实验组桑椹胚H19,Igf-2的表达分别是对照组的5.9和5.2倍(P < 0.01);实验组囊胚H19,Igf-2的表达分别是对照组的2.4和1.8倍(P < 0.01)。BSP测序法分析H19,Igf-2印迹控制区的甲基化水平发现,实验组桑椹胚、囊胚的甲基化率分别比对照组下降27.9%和20.9%(P < 0.01)。结果可见在高糖环境下胰岛素样生长因子1可部分降低H19/Igf-2印迹控制区DNA甲基化水平,使 Igf-2和H19基因表达增高,能拮抗高糖环境对小鼠早期胚胎发育的损伤。

Effect of insulin-like growth factor 1 antagonized high glucose on imprinted gene H19/Igf-2 in mouse preimplantation embryos

BACKGROUND:The previous studies have confirmed that 100 μg/L insulin-like growth factor 1 can antagonist apoptosis in vitro induced by 30 mmol/L glucose toxicity, it can relieve harm of high glucose on maternal/fetal. But the mechanisms and molecular mechanisms to improve the nutritional environment of the early embryo development are not clear.OBJECTIVE:To observe the effect of insulin-like growth factor 1 antagonist high glucose on mRNA expression and DNA methylation levels of the development-related imprinted genes H19/Igf-2 in mouse preimplantation embryos in vitro on the basis of preliminary work and starting from epigenetic prospect.METHODS:The diabetic mice models were established. The acquired early period 2-cell embryos were divided into experimental group and control group: the control group was high sugar adversity cultured in KSOM culture medium containing 30 mmol/L glucose, and the experimental group was treated with 100 μg/L insulin-like growth factor 1. Until growth to the blastocyst stage, the mRNA expression level of H19/Igf-2, and the DNA methylation level in imprinting control region of H19/Igf-2 were detected.RESULTS AND CONCLUSION:Real-time PCR analysis showed that the mRNA expression levels of H19 and Igf-2 in morulas were respectively 5.9 and 5.2 times as that of the control group (P < 0.01). Moreover, thus mRNA expression levels of H19 and Igf-2 in blastulas were separately 2.4 and 1.8 folds as that of the control group (P < 0.01). Bisulfite Sequencing PCR analysis demonstrated that the DNA methylation levels of H19 and Igf-2 in mouse morulas and blastulas of the experimental group were respectively declined by 27.9% and 20.9% than that of the control group (P < 0.01). The insulin-like growth factor 1 antagonist high glucose can partially reduce the DNA methylation level of H19/Igf-2 in imprinting control region in order to increase the mRNA expression levels of H19 and Igf-2, and it can antagonize the damage of high glucose during early mouse embryo development.