雌激素对Pax8介导的子代甲状腺发育及功能的影响机制研究

辅助生殖技术出生子代安全性日益受到关注。已证实促排卵使母体处于非生理性高雌激素状态，影响子代甲状腺功能，但机制不详。本项目在前期发现高雌致子代甲状腺功能异常及转录因子Pax8表达改变基础上，将深入研究雌激素对Pax8介导的甲状腺发育及功能的影响机制。拟利用宫内高雌小鼠模型，明确子代甲状腺不同发育阶段Pax8表达丰度及定位；利用mRNA-Seq及iTRAQ技术筛查甲状腺差异基因/蛋白，生物信息学分析重要通路候选因子，尤其Pax8相关分子网络；比较相应甲状腺功能指标及甲状腺激素合成代谢关键因子变化；建立过表达体系阐明Pax8对下游关键靶基因表达和活性影响；通过在体和体外模型检测高雌作用下Pax8启动子区及基因组DNA甲基化水平；利用胎鼠甲状腺细胞培养及CRISPR/Cas9特异性基因敲除细胞系，探索高雌通过调节Tet影响子代甲状腺细胞甲基化修饰的表遗传机制；为研究其他发育源性疾病开拓思路。

The safety of descendants of assisted reproductive technology has become a growing concern in the world. It has been confirmed that ovulation stimulation make the mother is in a non-physiological high estrogen status, affecting thyroid function in offspring. However, the mechanism is still unclear. We have previously found out that high estrogen induced thyroid dysfunction and altered the expression of transcription factor Pax8 in offspring. Here, we plan to further study the mechanism of the influence of estrogen on Pax8 mediated thyroid development and function. We are going to use the established intrauterine high estrogen mice model, detect the levels of Pax8 expression and location in diffferent developmental stages of thyroid in offspring; screen the differential genes/proteins of thyroid in offspring by mRNA-Seq and iTRAQ technology; predict the important pathway candidate factors by bioinformatic analysis, especially Pax8 related molecular network; compare corresponding thyroid function indicators and the expression levels of key moleculars of thyroid hormone synthesis and metabolism; establish overexpression system, demonstrate the effects of Pax8 on the expression and activity of downstream key targeted genes; with in vivo and in vitro models, analyze the methylation status of Pax8 promoter region and genomic DNA after high estrogen treatment; by in vitro culture of thyroid cells of fetal mice and the cell line with CRISPR/Cas9 specific gene knockout technology, explore the epigenetic mechanism of high estrogen affecting the methylation of thyroid cells in offspring by regulation of Tet; to provide new idears for studying other developmental origins of diseases.