Molecular determinants of thyroid cell functional plasticity

在许多物种中，甲状腺是正常发育和功能所必需的。甲状腺中的关键功能实体是血管滤泡单位(AFU)，它由特化的甲状腺上皮细胞和由内皮细胞围绕的血管网络组成。AFU中这些细胞类型的复杂特化(分化)对于甲状腺激素的产生是必不可少的。目前，关于甲状腺细胞和内皮细胞之间密切的结构联系的分子驱动因素缺乏信息。我们不知道甲状腺干细胞或分化的甲状腺细胞是否与内皮细胞沟通。周细胞是血管的支持细胞，其在AFU形成中的作用尚不清楚。我的研究项目调查了AFU分化的细胞和分子驱动因素，并探索了环境化合物如何干扰调节甲状腺AFU成熟的关键过程。我将集中讨论影响AFU细胞分化的两个机制：(1)血管内皮细胞的募集和分化是建立这个复杂的结构-功能单位的关键条件；(2)AFU中细胞核染色体端粒结构变化对AFU动态分化的影响。核端粒是哺乳动物染色体的保护端，其在细胞核内的组织结构随着细胞分化而改变。虽然端粒受到蛋白质的保护，但它们对氧化应激明显敏感。许多环境污染物都能引起细胞内的氧化应激。我的研究调查了氧化应激环境诱导剂引起的细胞核端粒组织的变化是否会损害AFU的细胞分化。方法：在我的实验室里，我们已经建立了甲状腺细胞的分离和培养、细胞分化的操作以及甲状腺上皮细胞和内皮细胞的二维和三维联合培养的技术。我将使用一种独特的三维(3D)细胞培养模型，涉及三种关键细胞类型来形成AFU：甲状腺细胞、内皮细胞和周细胞。我将在AFU中鉴定甲状腺(干细胞)细胞产生的刺激内皮细胞生长和血管形成的蛋白质，并将调查环境因素是否会干扰这一过程。我将利用选定的环境化合物来诱导端粒的氧化应激，从而改变端粒的组织结构。我将探索这些结构端粒变化对细胞分化为AFU能力的影响。这项工作利用超分辨率显微镜技术来可视化细胞核中端粒的结构变化。我将使用3D荧光成像和电子显微镜技术来检测AFU中的细胞类型和细胞分化标志物。我将利用功能测试来监测AFU的功能成熟度。新颖性/预期意义：这项研究将发现在甲状腺AFU功能分化中驱动内皮细胞募集的蛋白质，并将首次确立周细胞在这一基本生物学过程中的作用。这项工作将首次探索核端粒作为环境化合物的分子靶标的作用，并将由此产生的结构端粒变化与甲状腺细胞分化受损联系起来。我们的发现将启动新的发现，将环境信号与影响分化过程的核结构变化联系起来。

The thyroid gland is essential for normal development and function in many species. The key functional entity in the thyroid gland is the angio-follicular unit (AFU) which comprises specialized thyroid epithelial cells and a surrounding network of blood vessels lines by endothelial cells. The intricate specialization (differentiation) of these cell types in AFU is essential for thyroid hormone production. Currently, information is lacking on the molecular drivers for the close structural association between thyroid cells and endothelial cells. We don’t know whether thyroid stem cells or differentiated thyroid cells communicate to endothelial cells. The role for pericytes, supporting cells for blood vessels, in AFU formation is unknown. My research program investigates the cellular and molecular drivers for AFU diferentiation and explores how environmental compounds interfere with critical processes regulating thyroid AFU maturation. I will focus on two mechanisms which influence aspects of cell differentiation in AFU: (1) recruitment and differentiation of blood vessel endothelial cells as a key requirement in establishing this intricate structure-function unit, and (2) the influence of structural changes at chromosomal telomeres in the cell nucleus on the dynamic differentiation in AFU. Nuclear telomeres are the protective ends of mammalian chromosomes and their organization in the nucleus was shown to change with cell differentiation. Although telomeres are protected by proteins they are explicitly sensitive to oxidative stress. Many environmental contaminants can induce oxidative stress in cells. My research investigates whether changes to the telomere organization in the nucleus caused by environmental inducers of oxidative stress will impair cell differentiation in AFU. Methodology: In my lab, we have established techniques for the isolation and culture of thyroid cells, the manipulation of cell differentiation and the two- and three-dimensional co-culture of thyroid epithelial and endothelial cells. I will use a unique three-dimensional (3D) cell culture model involving three key cell types for AFU formation: thyroid cells, endothelial cells and pericytes. I will identify proteins produced by thyroid (stem) cells which stimulate endothelial cell growth and blood vessel formation in AFU and will investigate whether environmental factors interfere with this process. I will utilize selected environmental compounds to induce oxidative stress at telomeres with resulting changes to the telomere organization. I will explore the impact of these structural telomere changes on the ability of cells to differentiate into AFU. This work utilizes superresolution microscopy techniques to visualize structural changes at telomeres in the nucleus. I will use 3D fluorescence imaging and electron microscopy techniques to detect cell type and cell differentiation markers in AFU. I will use utilize functional tests to monitor functional maturation of AFU. Novelty/ expected significance: This research will discover proteins which drive endothelial recruitment in thyroid AFU functional differentiation and will establish, for the first time, a role for pericytes in this fundamental biological process. This work will, for the first time, explore the role for nuclear telomeres as molecular targets for environmental compounds and link the resulting structural telomere alterations to impaired cell differentiation in the thyroid. Our findings will initiate new discoveries linking environmental signaling with nuclear structural changes impacting on differentiation processes.