宫内高糖环境对雄性子代生殖细胞Igf2/H19基因印记的影响及其机制研究

妊娠期糖尿病（GDM）宫内高糖环境已证实在排除遗传因素后增加子代患糖尿病风险，宫内高糖环境还增加子二代小鼠糖耐量异常的风险，但此隔代效应机制仍有待研究。本实验组前期发现GDM子一代小鼠精子印记基因Igf2/H19表达异常，提示我们将发病关注点前移至配子发生期，研究对雄性子代生殖细胞印记重编程的影响。本项目将利用动物模型，检测GDM子一代雄性胎鼠不同阶段生殖细胞中Igf2/H19表达水平及相应差异甲基化区（DMR）甲基化状况；检测高糖环境下甲基转移酶（DNMT1，DNMT3a，DNMT3b）的水平，研究Igf2/H19 DMR甲基化改变的机制是否由DNMTs表达调控异常所致；进行体外细胞培养，应用转染及siRNA技术，纠正异常甲基化调控关键酶，比较分析改善甲基化修饰异常后对不良影响的消减作用；揭示宫内不良环境所致隔代效应的可能发生机制，为宫内高糖环境所致隔代发病效应实现源头控制提供理论基础。

Intrauterine hyperglycemia environment of gestational diabetes mellitus (GDM) has been confirmed to increase risk of diabetes in offspring beyond genetic factors. Intrauterine hyperglycemia environment also increase the risk of impaired glucose tolerance in the second filial generation of mice. However, the mechanism of transgenerational transmission remains to be studied. Our team previously found abnormal imprinted gene Igf2/H19 expression in the sperm of the first filial generation of GDM mice, which suggests us focus the concern of the onset of diseases at the phase of gametogenesis, investigating the effect on imprinting reprogramming of germ cells in male offspring. The project will use animal model, examine the level of Igf2/H19 expression and methylation status of differential methylation region (DMR) of germ cells of different stages in male fetus; detect the levels of DNA methyltransferase （DNMT1, DNMT3a，DNMT3b）in hyperglycemia environment to explore whether the mechanism of methylation change is associated with abnormal expression and regulation of DNMTs; use in vitro cell culture, apply technology of transfection and siRNA, correct abnormal key enzymes for methylation regulation; analyze the attenuation of adverse effect after improving abnormal methylation modification. We are in the hope of revealing the probable mechanism of transgenerational effect of intrauterine hyperglycemia environment, providing theory basis for controlling the transgenarational influence by intrauterine adverse environment at the source.