先天性甲状腺功能低下巴马猪模型构建及应用研究

Previous studies have shown high incidence of hypothyroidism, and epidemiological survey has indicated that the number of hypothyroidism patients in China has increased rapidly in recent years. Therefore, it is urgent to carry out the research on the pathogenesis and treatment of hypothyroidism. The animal models for human diseases have been widely used to unravel pathogenetic mechanisms of disease and for preclinical testing of drugs. In this study, we planned to reveal the association between DUOX2 mutation (D409G) and pig hypothyroidism, and further created the DUOX2 mutant pig which constructed by ENU chemical mutagenesis as the first large animal model for human congenital hypothyroidism. In addition, it was reported that hypothyroidism is often accompanied by chronic anemia (decrease in the total amount of red blood cells) which was also observed in our pig model. while, the molecular mechanism of the development of anemia in hypothyroidism patients remains unclear. To address this important issue, we intend to take full advantage of this animal model to reveal the molecular mechanism of the anemia of hypothyroidism by RNA-seq and Chip-seq (KLF9) analysis of bone marrow. Through the above research, this study aims to build a large animal model of human hypothyroidism, and the application of this animal model to reveal the molecular mechanism of the anemia of hypothyroidism may provide important targets for the clinical treatment of anemia and promote progress of translational medical research for human patients with hypothyroidism.

甲减是一种发病率较高的内分泌系统疾病，近年来尤其是我国的发病率呈上升趋势。虽然补充甲状腺激素可以治疗大部分的病人但仍有部分病人的治疗效果不佳，因此利用在甲状腺激素代谢模式上与人类更接近的大动物疾病模型开展人类甲减发病机制的研究及治疗方案的优化具有重要的意义。本项目利用ENU化学诱变已获得DUOX2 D409G巴马猪突变家系，初步的表型分析表明该家系具有甲状腺肿大，甲状腺激素降低，贫血等症状，与甲减病人类似。本项目计划对该突变体进行详细的表型分析，并解析DUOX2基因突变引起甲减的分子机制，以期构建能够较好模拟人类甲减的大动物模型；此外，针对甲减常伴有长期贫血这一危害较大但发病机制未明的重要问题，计划应用该模型重点解析甲减患者发生贫血的分子机制。通过以上研究，本项目旨在构建理想的人类甲减的大动物模型，并应用该模型解析人类甲减患者贫血的发病机制，为优化甲减的临床治疗和转化医学研究提供理论基础。

甲状腺功能减退（甲减）是一种发病率较高的内分泌系统疾病，近年来在我国的发病率呈上升趋势，对其发病机制的研究具有很大的价值和意义。大动物疾病模型在甲状腺激素代谢模式上与人类更加接近，因此通过大动物模型解析甲减的致病机制具有较大优势。本项目前期利用ENU化学诱变技术已获得DUOX2 D409G/ D409G巴马猪突变体，表型分析表明该突变体具有甲状腺肿大，甲状腺激素降低，贫血等症状，总体能够模拟人类甲减的疾病表型。在此基础之上，本项目利用DUOX2 D409G/D409G突变体巴马猪，深入解析其致病机制，取得重要研究进展：首先，通过CRISPR/Cas9技术制备DUOX2基因双等位基因敲除猪，证实DUOX2 D409G/D409G为致病突变，表明DUOX2 D409G/D409G突变体猪可作为模拟人类甲减的大动物模型；其次，证实D409G突变影响DUOX2 mRNA的可变剪切，从而导致双氧化酶2的活性和功能显著降低，最终导致严重的甲状腺激素合成障碍，解析了DUOX2 D409G/D409G的致病分子机制。再者，发现“TR-KLF9”信号轴是甲状腺激素调控造血系统发育的关键靶点，并解析“TR-KLF9”调控红细胞和免疫细胞发育的分子机制。本项目的研究成果具有重要价值，一方面构建和提供了可作为人类甲减临床和基础研究使用的大动物模型，另一方面本项目发现的“TR-KLF9”信号轴及其作用机制，为甲减的临床治疗和转化医学研究提供重要理论基础。