甲状腺滤泡内甲状腺球蛋白负反馈调节滤泡功能作用与机制的研究

The follicles are the structural and functional units of the thyroid. Thyroid stimulating hormone (TSH) is known as the master regulator to stimulate the follicles to biosynthesize thyroid hormone. Thyroglobulin (TG), the precursor of thyroid hormone, is accumulated in the follicular colloid. Our research group has reported that TG can counteract the TSH action to suppress both the expression and function of key molecules necessary for the biosynthesis of thyroid hormone, in a concentration-dependent manner. Based on evidence, we have proposed “a negative-feedback effect of follicular TG on the follicular function”, and “a follicular model” to explain the maintenance of follicular homeostasis. However, whether TG actually affects thyroid hormone secretion in the thyrocytes, and by which mechanism it exerts its effect, remains unclear. In this project, we shall exam whether TG suppresses thyroid hormone secretion using a novel culture system of primary human thyrocytes; we shall determine whether the follicular TG contents are negatively related to the expressions of thyroid-functional molecules in human thyroid tissues; we shall investigate whether TG exerts its effect via inhibiting TSH signaling pathway or another signaling pathway that was screened out in our previous study; we shall also explore a novel apical receptor that potentially mediate the negative-feedback effect of follicular TG. We believe this project will significantly widen our understanding about the regulation of thyroid function.

甲状腺滤泡是甲状腺最基本的结构功能单位，促甲状腺激素（TSH）是促进滤泡合成甲状腺激素功能的“总司令”。甲状腺球蛋白（TG）是甲状腺激素前体，贮存于甲状腺滤泡腔内。申请者课题组发现，TG可拮抗TSH作用，抑制甲状腺激素合成“流水线”上各功能分子的表达及功能，提出了“甲状腺滤泡内TG负反馈调节滤泡功能”学说。然而，前期实验中常用的大鼠甲状腺细胞无法用于测定TG对细胞产生甲状腺激素的影响，且TG负反馈作用的分子机制尚不明。本课题，通过建立人甲状腺细胞离体极性培养模型，直接检测TG对细胞产生甲状腺激素功能的影响；观测人甲状腺滤泡内TG含量与上皮细胞功能分子表达量的负相关性；明确TG是否通过抑制经典的TSH信号通路，或通过调节（由前期研究筛选所得）非TSH通路，以发挥其负反馈调节作用；探索滤泡内腔侧可能存在的介导TG负反馈调节作用的未知受体。本研究将为我们理解甲状腺功能稳态及功能异常提供新思路。

甲状腺最主要的生理功能即合成并分泌甲状腺激素。甲状腺滤泡内的甲状腺球蛋白（TG）可拮抗促甲状腺素（TSH）作用，抑制甲状腺细胞中甲状腺激素合成“流水线”上各功能分子的表达及功能。据此，我们前期提出了滤泡内TG负反馈调节甲状腺细胞合成甲状腺激素功能的假说。然而，由于甲状腺细胞系在体外并不具备分泌甲状腺激素的功能，在传统的单层甲状腺细胞培养体系中无法测定TG对细胞实际分泌甲状腺激素的影响，且TG负反馈作用的分子机制尚不明。本课题，我们使用手术中获得的正常人甲状腺组织分离出原代人甲状腺细胞，将其培养在transwell内室孔膜上。待细胞长满后，内外室更换成不同成分的培养基，从而模拟甲状腺细胞在体内的极性状态。为了检测原代甲状腺细胞体外分泌甲状腺激素的水平，我们使用了无血清培养基，以排除含血清培养基中外源性激素的干扰。我们在该人原代甲状腺细胞极性模型中，证实了离体培养的原代人甲状腺细胞可以在体外分泌甲状腺激素。原代甲状腺细胞分泌激素的水平与供体的年龄呈负相关。同时我们观测到，在transwell内室添加外源性TG，可抑制甲状腺细胞中功能分子的表达水平。并且，通过蛋白质谱鉴定证实，外源性TG处理后，甲状腺细胞内源性TG含量明显下降；二代测序的结果同样证实，外源性TG处理后，甲状腺细胞中甲状腺激素合成“流水线”上各功能分子的表达显著下调。该些结果为滤泡内TG负反馈调节甲状腺细胞合成甲状腺激素功能的假说提供了进一步的证据。二代测序及蛋白质谱检测到的TG处理前后的表达差异分子，进一步通过生物信息学分析，筛选出显著性排名前20的信号通路，是今后深入研究TG负反馈作用分子机制的重要依据。