Thyroid hormone conversion in vitro and ex vivo studies

本实验室目前正在研究2型脱碘酶基因常见多态在体外和体外的功能活性。为了实现这一目标，我们建立了基于细胞培养的2型脱碘酶表达系统。酶活性测定采用标准生物化学方法，蛋白质/蛋白质和核酸/蛋白质相互作用采用免疫沉淀法和迁移率改变法检测。DIO2基因5UTR区的一个常见的多态(258A/GDIO2)与循环中的T3/T4比值的改变有关，这表明体内该酶的活性增加导致反应平衡的改变。我们的体外和体外数据与其他基因型/表型相关研究一致，表明258A/G DIO2变异通过取代假定的阻遏因子而诱导基因转录增加。目前的研究目的是确定与该基因多态相互作用的抑制因子。对数据的初步分析表明，有多个候选转录因子。我们目前的努力旨在描述压迫者的特征。 前脂肪细胞的分化和培养。在2010财年，我们集中精力开发了一种从脂肪组织活检过程中获得的基质细胞再分化为人类脂肪细胞的系统。该系统允许以受控的方式测试特定的基因类型对脂肪组织功能的影响，而与采样时受试者的新陈代谢状态无关。我们目前正在使用这个实验模型来表征甲状腺激素在脂肪细胞分化中的作用。进一步的努力旨在描述该系统在整个分化过程中的转录模式，特别是棕色脂肪特异性基因的表达。 滤泡甲状腺细胞原代培养中的2型脱碘酶测定。与Gershengorns博士团队(NIDDK-CEB)开展的合作工作已经开发出一种可靠的方法，用于测量甲状腺细胞原代培养中的2型脱碘酶活性，作为TSH-cAMP途径的读出。该系统已成功地用于检测TSH受体激动剂的活性。 开发一种基于细胞的系统，以表征调节2型脱碘酶活性的物质的影响。我们目前正在开发稳定表达2型脱碘酶的细胞系和甲状腺激素反应元件(TrE)驱动的报告结构，以评估调节2型脱碘酶活性的小物质的活性。

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.