Thyroid hormone conversion in vitro and ex vivo studies

甲状腺激素体外和离体转化研究

• 批准号: 8157995
• 负责人: FRANCESCO S CELI
• 金额: $ 64.85万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157995
• 关键词: Adipocytes; Adipose tissue; Cyclic AMP; Data; Data Analyses; Development; Elements; Equilibrium; Genes; Genotype; Goals; Homeostasis; Hormone Responsive; Hormones; Human; Immunoprecipitation; In Vitro; Pathway interactions; Peripheral; Process; Stromal Cells; Thyroid Hormones; Variant; base; in vivo

Our laboratory is currently characterizing in vitro and ex vivo the functional activity of common polymorphisms of type-2 deiodinase gene. In order to achieve this goal, we have established a cell culture-based type-2 deiodinase expression system. Standard biochemistry methods are utilized for the enzymatic activity assay; protein/protein and nucleic acids/protein interactions are tested by immunoprecipitation and mobility shift assay. A common polymorphism in the 5UTR region of the DIO2 gene (258 A/G DIO2) has been associated with a shift in the ratio of circulating T3/T4, suggesting an increased in the activity of the enzyme in vivo leading to a shift of the reaction equilibrium. Our in vitro and ex vivo data, consistent with others genotype/phenotype association studies, indicate that the 258 A/G DIO2 variant induces an increase in the transcription of the gene by displacing a putative repressor. Current efforts are aimed to characterize the putative repressor factor interacting with the polymorphism. Preliminary analysis of the data has indicated multiple candidate transcription factors. Our current efforts are aimed to the characterization of the repressor. Pre-adipocyte differentiation and culture. During FY 10 we have focused our effort in developing a system of re-differentiation of human adipocytes from stromal cells obtained during adipose tissue biopsy. This system allows to test in a controlled fashion the effects of specific genotypes on adipose tissue function independently of the metabolic status of the subject at the time of the sampling. We are currently using this experimental model to characterize the role of thyroid hormones in the differentiation of the adipocytes. Further effort is directed toward characterizing the transcriptional pattern of this system throughout the differentiation process with a particular focus on the expression of brown-fat specific genes. Type-2 deiodinase assay in primary culture of follicular thyroid cells. Collaborative work carried out with Dr. Gershengorns group (NIDDK-CEB) has led to the development of a reliable assay for the measurement of type-2 deiodinase activity in primary cultures of thyroid cells as a read-out of TSH-cAMP pathway. This system has been successfully utilized to test the activity of agonists of the TSH receptor. Development of a cell-based system to characterize the effects of substances modulating the type-2 deiodinase activity. We are currently developing cell line stably expressing type-2 deiodinase and a Thyroid Hormone Responsive Element (TRE)-driven reporter construct in order to evaluate the activity of small substances modulating the type-2 deiodinase activity.

我们的实验室目前正在体外表征体外和体内，是2型去二酸酶基因的常见多态性的功能活性。为了实现这一目标，我们已经建立了基于细胞培养的2型去二酸酶表达系统。标准生物化学方法用于酶活性测定；蛋白/蛋白质和核酸/蛋白质相互作用通过免疫沉淀和迁移性转移测定测试。 DIO2基因5UTR区域的常见多态性（258 A/G DIO2）与循环T3/T4的比率变化有关，这表明体内酶的活性增加，导致反应平衡的转移。我们的体外和离体数据与其他基因型/表型关联研究一致，表明258 A/G DIO2变体通过取代推定的阻遏物来诱导基因转录的增加。目前的努力旨在表征与多态性相互作用的假定阻遏因子。对数据的初步分析表明多个候选转录因子。我们目前的努力旨在表征阻遏物。 前脂肪细胞的分化和培养。在10财年期间，我们将精力集中在开发从脂肪组织活检期间获得的基质细胞的人脂肪细胞重新分化的系统。该系统允许以受控的方式测试特定基因型对脂肪组织在抽样时的代谢状态的影响。我们目前正在使用该实验模型来表征甲状腺激素在脂肪细胞分化中的作用。在整个分化过程中，进一步的努力是针对表征该系统的转录模式的，特别着眼于棕色特异性基因的表达。 卵泡甲状腺细胞的原代培养物中的2型去二酸酶测定。与Gershengorns Group（NIDDK-CEB）进行的协作工作已导致开发可靠的测定法，以测量甲状腺细胞的原发性培养物中2型去二酸酶活性，作为TSH-CAMP途径的读出。该系统已成功用于测试TSH受体激动剂的活性。 开发基于细胞的系统，以表征调节2型去二酸酶活性的物质的影响。目前，我们正在开发稳定表达2型去二世酶的细胞系和甲状腺激素反应元件（TRE）驱动的报告基因构建体，以评估调节2型脱碘酶活性的小物质的活性。