动物造模,甲减猪模型 z-bio 2021-05-27 426

　　Congenital hypothyroidism (hypo hypothyroidism) is an endocrine disorder caused by insufficient secretion of thyroid hormones. The global incidence of newborns is as high as 1:1 400-1:2800. Clinically, 20% to 60% of patients with hypothyroidism have symptoms such as anemia and immunodeficiency, but the molecular mechanism is still unclear. Thyroid hormone replacement therapy is a common treatment for patients with hypothyroidism, but related reports have shown that in some patients with severe hypothyroidism and anemia, thyroid hormone cannot be completely cured. This indicates that severe thyroid hormone deficiency can cause damage. It cannot be undone completely. Appropriate animal models are essential for analyzing the causes of diseases and developing new drugs and treatments. Compared with large animals such as mice, pigs and other model animals, not only the organ size, physiological and biochemical characteristics are similar to humans, but also the metabolism and immunity of thyroid hormones are similar to humans. More importantly, porcine thyroid gland can be used as one of the main sources of natural thyroid hormone, and pig is a good animal model for simulating human hypothyroidism.

　　Under the auspices of Chinese Academy of Sciences scholars Meng Anming and Zhou Qi, the Institute of Zoology, Chinese Academy of Sciences took the lead in establishing the "China Pig Chemical Mutagenesis Alliance" using ENU chemical mutagenesis technology. Many pig disease models and mutants were effectively created, and recessive families of congenital hairless mutants were discovered. In this study, phenotypic analysis revealed that these mutants exhibited severe hypothyroidism similar to humans. Family-based whole-genome linkage analysis and whole-genome sequencing confirmed that the disease-causing gene DUOX2 has a point mutation. In other words, the conversion of c.1226A\→G will change the corresponding amino acid sequence D409G. The CRISPR/Cas9 system was used to knock out the corresponding region of the normal pig gene, indicating that the phenotype of the mutant matched the phenotype obtained by ENU mutagenesis, making DUOX2 pathogenic to the family, and further confirmed that it is the gene.

　　DUOX2 and its maturation factor DUOXA2 produce H2O2, thyroid thyroid hormone synthesis uses hydrogen peroxide as a substrate for thyroid peroxidase. Peroxidase is involved in the iodination process of thyroglobulin tyrosine residues and iodinated tyrosine. This step is thyroid hormone. This is the rate-determining step of the synthesis. The D409G mutation is located in the peroxidase-like domain of DUOX2, which greatly reduces the stability of the DUOX2 protein. Both in vivo and in vitro experiments have confirmed that mutations can reduce the production of H2O2 in the thyroid. In short, the D409G mutation destroys the stability of the DUOX2 protein, affects the production of H2O2 in the thyroid and leads to a reduction in thyroid hormone synthesis.

　　A detailed study of the phenotype of the pig model of hypothyroidism also showed severe symptoms of anemia and immunodeficiency. Transcriptome sequencing of thymus tissues significantly reduced the expression of the important transcription factor KLF9 in mutant thymus tissues, thereby further studying the molecular mechanisms between hypothyroidism, anemia and immunodeficiency. Thymus is the main organ producing T lymphocytes. It is speculated that KLF9 may affect the production of hematopoietic cells. In vivo and in vitro experiments have confirmed that the pig KLF9 gene is directly regulated by the thyroid hormone receptor in a thyroid hormone-dependent manner in hematopoietic cells. In order to see if KLF9 (a gene located directly downstream of the thyroid hormone receptor) affects the development of hematopoietic function, after knocking out Klf9, mature red blood cells and T lymphocytes, I further used the zebrafish model and I confirmed it The reduction. The possible molecular mechanism is that Klf9 affects the maturation of red blood cells and the production of T lymphocytes by regulating the process of apoptosis and division. This study created the first porcine hypothyroidism model and was the first to discover that KLF9 mediates thyroid hormone receptors to regulate hematopoiesis and immune cell development. This reveals the root cause of anemia and immunodeficiency. Hypothyroidism provides theoretical support for in-depth medical research on human hypothyroidism, and also provides a theoretical basis for new pig breeds with high immunity and healthy pig industry.