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

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.

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。

研究背景：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超生理性高雌激素状态，影响子代甲状腺功能，但机制不详。我们已证实，促排卵所致的母体非生理性的甾体激素状态使得胎儿发育处于高雌激素的宫内环境中，并利用前期建立的宫内高雌激素小鼠模型，发现高雌激素可能通过干扰甲基化修饰影响子代甲状腺转录因子Pax8 表达，从而可能导致子代甲状腺功能异常。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，拟利用宫内高雌小鼠模型及体外实验，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，为研究其他发育源性疾病开拓思路。.研究内容：1、宫内高雌对子代甲状腺转录因子Pax8 表达和定位的影响；2、宫内高雌致子代甲状腺功能异常的重要分子通路；3、Pax8 过表达对下游关键靶基因转录调控与募集的影响；4、宫内高雌对子代甲状腺DNA甲基化的影响及机制。.研究结果：1、高雌组子代小鼠甲状腺激素水平出现异常改变，甲状腺Pax8 表达显著上调；2、重点关注了与甲状腺发育和功能密切相关的基因Tpo，Tg，Nis，Pax8和Titf1，发现Pax8和Tpo表达显著上调；3、高雌组子代小鼠Pax8基因启动子区域CpG岛甲基化水平显著低于对照组，甲基化调控酶Dnmt3a和Mbd1基因的表达要显著低于对照组；4、体外实验中，雌激素能够浓度依赖性地促进甲状腺滤泡细胞系的增殖，以及上调pax8基因和下调Dnmt3a基因；5、此外也研究了lncRNA 在甲状腺细胞直接暴露于高雌激素环境下是否可能对影响甲状腺细胞的表观遗传修饰，进而影响其生物学功能。.科学意义：宫内高雌激素影响子代甲状腺功能相关基因表达进而导致甲状腺激素分泌异常，甲状腺细胞甲基化修饰改变可能是机制之一。