叶酸通过DNA甲基化途径对脑发育表观遗传学作用机制的研究

The aim of this study is to explore the epigenetic effects of folic acid on brain development by DNA methylation. The relationship between folic acid, DNA methylation and brain development will be studied by the three experimental levels and three approach experiments. The three experimental levels are animal reproductive study, neural stem cells (NSCs) in vitro, and population cohort study. The brain development, methionine circulating metabolites and DNA methylation will be detected. Pregnant rats were randomly assigned to 3 groups: folic acid deficient, folic acid supplement for short period, and folic acid supplement for whole period of gestation and before pregnancy. And then pups were randomly assigned to 3 parts: one part pups were killed to get brain tissue, one part pups used to culture hippocampal NSCs in vitro, the last part pups feed till three month. Cohort study follows up women and their infants for three years. The women and their infants will be divided to three groups according to the situation of folic acid supplement during pregnancy: supplement folic acid during whole gestation and before pregnancy, supplement folic acid only a short time, and never supplement folic acid. Brain development, learning and memory abilities of rat offspring and infants will be examined. The levels of folate, S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, 5-methylcytosine, DNA methyltransferase activity and genes experession of rat brain tissue and blood and in the umbilical cord blood of women, the proliferation and differentiation of NSCs will be detected. The levels of DNA methylation will be assessed by MeDIP-seq, and differentially methylated regions will be found. The study will elucidate the underlying mechanism of the effects of folic acid deficiency and the different way of folic acid supplement on brain development, and demonstrate the relationship of folate, DNA methylation and brain development. The molecular mechanisms of the epigenetic effects of folic acid on brain development by DNA methylation will be provided. A scientific guideline for folic acid supplement in women will be suggested.

本研究通过动物生殖实验、体外神经干细胞（NSCs）培养及人群队列研究三个层次，脑发育测量、蛋氨酸循环代谢产物、DNA甲基化基因三条途径，探讨叶酸-DNA甲基化-脑发育三者之间的关系。采用大鼠生殖实验，分为孕期叶酸缺乏、短期服用和全程服用叶酸组，每组大鼠产生的同窝子代新生鼠分为三部分：子代新生鼠脑组织；子代新生鼠海马NSCs体外培养；子代新生鼠将继续饲养至3月龄，然后进行检测。人群研究将孕产妇和婴幼儿分为孕前与孕期全程补充叶酸组、怀孕前后短期补充叶酸组以及孕前与孕期均未补充叶酸组，开展为期3年队列研究。检测大鼠子代大脑发育、学习记忆、NSCs增殖分化及婴幼儿神经发育。 检测大鼠子代大脑组织、NSCs、血液及孕妇脐带血中叶酸、蛋氨酸循环产物、5-甲基胞嘧啶、甲基转移酶活性及其基因表达；检测全基因组甲基化状态，找出差异基因，并进行验证。提出叶酸通过DNA甲基化途径对脑发育表观遗传学的作用机制。

孕期的营养状况对生命早期乃至生命全程健康状况的影响受到广泛关注。本研究开展大鼠生殖实验和母婴出生队列研究，通过动物生殖实验、体外神经干细胞（NSCs）培养及人群队列研究三个层次，脑发育测量、蛋氨酸循环代谢产物、DNA甲基化三条途径，探讨叶酸-DNA甲基化-脑发育三者之间的关系。出生队列研究结果显示，母亲孕期服用叶酸补充剂能够促进1月龄和24月龄子代神经发育。母亲孕期叶酸补充通过降低血清同型半胱氨酸（Hcy）和炎性因子水平，最终促进1月龄婴儿神经发育。不同MTHFR基因型的母亲孕中期血清叶酸、Hcy、S-腺苷甲硫氨酸（SAM）、S-腺苷同型半胱氨酸（SAH）和5-甲基胞嘧啶（5mC）水平差异无统计学意义（P>0.05）。大鼠生殖实验结合体外NSCs培养验证孕期叶酸影响子代神经发育的作用机制，结果显示，孕期叶酸补充可促进子代大鼠早期感觉-运动系统发育，提高青少年期和成年期的空间学习记忆能力，且长期补充叶酸效果优于短期补充叶酸。孕期叶酸缺乏使子代鼠脑组织海马区超微结构发生一系列病理改变，增加子代鼠脑海马区和皮层区神经细胞凋亡；而孕期补充叶酸可增加新生鼠海马区神经元和增殖的NSCs数量，促进皮层突触相关蛋白表达。MeDIP-chip高通量筛选甲基化差异富集基因（DEGs）显示孕期叶酸摄入可能通过甲基化表观遗传调控子代神经细胞发育、神经元间信号转导及突触可塑性等过程，但在不同性别中调控机制不同。研究结果提示合理补充叶酸可以促进子代的神经发育，孕期叶酸缺乏会对子代神经发育产生不利影响。研究结果为育龄人群合理补充叶酸促进子代的神经发育提供科学依据，具有重要的理论意义和实用价值。