【作者】 黄惠斌；

【导师】 林玲；

【作者基本信息】 福建医科大学 ， 内科学， 2010， 博士

【摘要】 碘对甲状腺功能的影响表现为循环碘及甲状腺内部有机碘储备状况改变了甲状腺自身的碘代谢行为及其对垂体TSH的敏感性。这种作用不依赖于垂体TSH,其目的是维持合适碘池、稳定甲状腺功能,属于甲状腺功能的自我调控作用。甲状腺激素的合成与分泌是一个连续而复杂的生理过程,主要涉及钠共转运体(NIS)、甲状腺髓过氧化物酶(TPO)、甲状腺球蛋白(TG)、及促甲状腺素受体(TSHr)等甲状腺特异蛋白及这些蛋白表达的主要转录调控因子TTF-1、TTF-2、PAX8的表达及活性。甲状腺内外碘的变化,通过各种复杂的机制,最终导致甲状腺转录因子及甲状腺特异蛋白的变化,实现甲状腺功能的自我调控。甲状腺功能调控的研究,需选择合适的实验模型。动物水平的研究由于影响因素众多,不易阐明某种因素的具体作用及机制;体外条件下,多数甲状腺细胞,呈单层细胞生长,无法形成滤泡结构,缺乏完整的激素合成、储备及分泌功能,均不是甲状腺功能调控研究的最佳选择。体外重构甲状腺滤泡,克服了上述二种模型的不足,既脱离了体内复杂的调控环境,又兼备完整的功能,是甲状腺功能调控研究理想实验模型。我们探索体外重构猪甲状腺滤泡的诱导方法,从甲状腺滤泡的形态、功能及甲状腺特异蛋白表达三方面证实体外重构猪甲状腺滤泡模型构建成功。在此基础上,利用Wenstern-blolt、RealTimeRT-PCR、ELISA、RIA等方法及免疫荧光、激光共聚焦显微镜观察、创新的甲状腺滤泡腔TG提取等技术,对高碘(HI)及低碘(LI)下甲状腺特异蛋白,甲状腺转录因子、甲状腺激素分泌、滤泡腔TG的碘化度进行深入研究,探讨碘对甲状腺功能的调控作用及分子机制,现将结果总结如下:一.猪体外重构甲状腺滤泡的构建及鉴定:1.猪体外重构甲状腺滤泡的构建方法:依照下列要点:①猪甲状腺低胰蛋白酶(0.1%)消化。②震荡法分散细胞。③1.5-3X106/L高密度、均匀接种。④接种头3天时在培养基中加入1mIU/mlTSH及15%的胎牛血清。⑤滤泡形成后改胎牛血清为10%小牛血清。经过3天的培养,即可形成了大量球状或半球状的重构甲状腺滤泡。2.体外重构甲状腺滤泡的形态学鉴定:用普通倒置显微镜可以观察到形成的圆圈样结构及细胞团,直径约10-100um;相差倒置显微镜下,圆圈样结构为半球状滤泡的贴壁面,穹窿顶由细胞构成;免疫荧光染色,利用激光共聚焦显微镜的“光学切片”作用下可以清晰地观察到细胞团是由周边的甲状腺细胞及内部的滤泡腔构成。3.体外重构甲状腺滤泡的功能学鉴定:①重构滤泡组上清液中有FT3、FT4的分泌,滤泡腔有TG的储备,而单层细胞组几乎未能检测到FT3、FT4分泌及TG储备。②重构滤泡组:高碘干预甲状腺特异蛋白NIS、TG、TPO、TSHr及甲状腺转录因子TTF-1、PAX8mRNA的表达高于低碘干预组。单层细胞组:高碘干预组上述指标表达均低于低碘干预组。二.以体外重构猪甲状腺滤泡为实验模型,研究碘对甲状腺功能的调控作用:1.高碘干预组甲状腺激素合成相关基因NIS、TPO、TGmRNA表达显著高于低碘组。2.高碘干预组培养上清液中FT3、FT4分泌显著多于低碘组。三.碘对甲状腺功能调控作用的分子机制研究:1.高碘干预组TSHr、TTF-1、PAX8mRNA表达及蛋白合成显著高于低碘组。2.①高碘干预组甲状腺滤泡腔TG碘化度高于低碘组;②高碘+ MMI(甲硫咪唑)组较单纯高碘组滤泡腔TG的碘化度显著下降;③高碘组TSHr、TTF-1、PAX8mRNA表达及蛋白合成较低碘组及高碘+MMI显著上调。④高碘+MMI组较MMI+高碘组TSHr、TTF-1、PAX8mRNA表达有升高的趋势。(高碘+MMI组:先高碘干预36h,再加入MMI;MMI+高碘组:先MMI干预36h,再加入高碘.即高碘、MMI干预顺序不同。)3.①PKA阻断组:TTF-1、TSHrmRNA表达及蛋白合成较对照组显著上调。②IP3阻断组:TTF-1、TSHrmRNA表达及蛋白合成较对照组显著上调。③PKA阻断组及IP3阻断组:NIS的表达较对照组上调。④PKA阻断组:NIS2的表达较对照组上调;IP3阻断组:NIS2的表达较对照组下调。结论:1.保持细胞膜的完整性、高密度均匀接种、增加细胞的接触、粘附、聚集的机会和能力,是体外重构甲状腺滤泡形成的关键;避免使用胎牛血清是防止甲状腺滤泡结构蜕变为单层结构,维持甲状腺滤泡形态的重要因素。2.体外重构甲状腺滤泡脱离了体内复杂环境,又具备完整的甲状腺功能,是甲状腺功能调控作用及机制研究的理想实验模型,可用于观察甲状腺滤泡形成过程,探讨甲状腺滤泡结构、滤泡内容物对甲状腺功能的调控作用。3.高碘组甲状腺激素合成相关基因NIS、TG、TPO高表达,培养上清液FT3、FT4高分泌,甲状腺激素的合成及分泌功能处于活跃状态,在滤泡层面上,碘对甲状腺激素合成及分泌功能具有促进作用。4.碘离子、MMI影响滤泡腔TG的碘化,其本身对TSHr及TTF-1的表达无直接作用;TSH/TSHr信号的CAMP/AKP及磷脂酶C/IP3途径对TTF-1、TSHr的表达亦无上调作用,TTF-1、TSHr高表达是滤泡腔TG高碘化的直接结果。5.碘离子提高了滤泡腔TG的碘化度,通过调控TTF-1,进而上调TSHr的表达,增强了甲状腺滤泡对中枢TSH/TSHr信号的敏感性,是其功能自我调控的重要途径之一。6. TG高碘化时,TSHr表达上调,滤泡对中枢信号敏感,促进了NIS、TPO、TG的表达及激素的分泌。是其功能活跃的原因之一。7.TSH/TSHr信号的不同传导途径对NIS及NIS2具有不同的调控作用,提示在NISmRNA剪接水平上中枢信号参与NIS不同亚型表达的调控。综上,我们发现:在重构滤泡这一层面上,碘影响滤泡腔TG的碘化度,通过调控TTF-1,进而上调TSHr的表达,改变了滤泡对中枢TSH/TSHr信号的敏感性。TG的碘化程度不同,滤泡对中枢TSH/TSHr信号敏感性存在差异,是甲状腺适应内外碘环境的变化,实现功能自我调控的重要途径之一;TG高碘化时,TSHr表达上调,滤泡对中枢信号敏感,促进了NIS、TPO、TG的表达及甲状腺激素的分泌。在滤泡水平上,碘通过上述机制有效促进甲状腺激素的合成与分泌。近10余来,高碘相关的甲状腺功能异常、自身免疫性甲状腺炎及甲状腺肿瘤发病率呈升高趋势。碘如何调控甲状腺功能,目前知之甚少,我们的工作为该领域的研究提供了科学实用的实验模型、拓宽了研究思路,奠定了一定的方法学基础,对探讨甲状腺疾病的发病机制及科学制定补碘策略具有重要理论和现实意义。更多还原

【Abstract】 The reserve of organic thyroid iodine and its circulation change the metabolism of thyroid and its sensitivity to pituitary TSH, which reflects the effects of iodine on thyroid function and is independent of pituitary TSH. This mechanism is a part of the self-regulation of thyroid function, which is conducive to keeping suitable iodine content and the stability of thyroid function.The synthesis and secretion of thyroid hormone are durative and complicated physiological process which relate to the expressions of thyroid-specific proteins including the sodium/iodide cotransporter (NIS), thyroid peroxidase (TPO), thyroglobulin (Tg) and thyroid stimulating hormone receptor (TSHr) as well as their transcriptional regulators, such as TTF1, TTF-2 and PAX8. The changes of internal and external iodine concentration bring about the alteration of the expressions of thyroid transcriptional factors and thyroid-specific proteins through various complicated mechanisms, which finally results in the self-regulation of thyroid function.The proper experimental model is a key point for the research on the regulation of thyroid function. Research in animals is affected by many factors and is unable to elucidate the detailed functions and mechanisms of some element; most thyroid cells cultured in vitro grow as a monolayer and cannot form the follicle structure, they do not possess the ability of hormonal synthesis, reserve and secretion, which are unsuitable for research on thyroid functional regulation. The recombinant thyroid follicle (RTF) is an ideal experimental model for thyroid functional regulation because it both breaks from the complex regulatory conditions in vivo and possesses the integral functions.We probed the methods of inducing swine recombinant thyroid follicle in vitro, and the RTF model was confirmed to be successful through identifying the morphology and function of thyroid follicle and the expression of thyroid-specific proteins. A series of methods including Western-blotting, realtime RT-PCR, ELISA, RIA, immunofluorescence, laser scanning confocal microscope and the techniques for extraction of thyroid follicular TG were employed to investigate the secretion of thyroid hormone, iodinated extent of follicular TG and the expressions of thyroid-specific proteins and thyroid transcriptional factors under the stress of high or low concentration of iodine. We aimed to elucidate the molecular mechanisms of iodine-regulated thyroid function. The results are as follows:(一)Establishment and identification of swine recombinant thyroid follicle①The methods of establishing swine recombinant thyroid follicle in vitro are briefly introduced as follows: thyroid tissues of swine were digested with 0.1% typsin and vibrated to disperse cells. Cells were maintained in RPMI-1640 medium supplemented with 15% fetal calf serum containing 1 mIU/mlTSH at the density of 1.5-3x106/L and the next day 10% calf serum replaced the fetal calf serum. After cultured 3 days thyroid cells accumulated together to form recombinant thyroid follicle.②Morphological identification of recombinant thyroid follicle Thyroid cells accumulated together to form a circular structure with diameter of 10 to 100 um under the inverted microscope. Observation of phase-contrast microscope revealed the circular structure was the surface of hemisphere follicle closing to the coverslip and the fornix was established with lots of cells. Laser confocal scanning microscope observation revealed that the cell aggregate consisted of thyroid cells and the inner follicle, which could be clearly visualized after immunofluorescence staining.③Functional identification of recombinant thyroid follicleIn the supernate of RTF group FT3 and FT4 were secreted and TG was reserved in the follicle, while all of them were not detected in the supernate of MC group.④Under the stress of high concentration of iodine the expressions of thyroid-specific proteins, NIS, TG, TPO, and TSHr, and the mRNA levels of thyroid transcriptional factors, TTF-1 and PAX8 were all up-regulated in RTF group, whose levels were all down-regulated in MC group.(二)Study the role of iodine on the regulation of thyroid function based on the in vitro experimental model of swine recombinant thyroid follicle①The mRNA levels of NIS, TPO and TG, which were related to the synthesis of thyroid hormone, were all up-regulated in HI group, while were down-regulated in LI group.②The secretion of FT3 and FT4 were enhanced in the supernate of HI group medium, while were significantly decreased in LI group supernate.(三)The molecular mechanism of iodine on the regulation of thyroid function①In group HI, the mRNA levels of TSHr, TTF-1 and PAX8 were all up-regulated as well as their protein synthesis.②The iodinated extent of follicular TG in group HI was relative higher than that in other groups, and it was decreased in group“HI+MMI”; when compared with“MMI+HI”group the iodinated extent of TG in group“HI+MMI”significantly rose.(“MMI+HI”group and“HI+MMI”group were dealt with a different procedure; cells in“HI+MMI”group were first treated with high concentration of iodine for 36 h and then MMI while cells in group“MMI+HI”were treated with a reverse sequence.)When follicular TG was highly iodinated, the mRNA levels of TSHr, TTF-1 and PAX8 were markedly improved as well as their protein synthesis.④The mRNA levels of TTF-1 and TSHr in group of H89 (inhibitor of PKA) and LY294002 (inhibitor of IP3) were both significantly enhanced as well as their protein synthesis;⑤The expression of NIS in group of H89 (inhibitor of PKA) was down-regulated while NIS2 (the subtype of NIS) was up-regulated; while in group of LY294002 the expression of NIS and NIS2 were up-regulated and down-regulated, respectively.Conclusions:1. Keeping the integrity of cell membrane, seeding at high density and increasing the opportunities of cell aggregation, adhesion and contact were the key points of successfully establishing recombinant thyroid follicle in vitro; one very important element was avoid using fetal bovine serum which could drive cells forming thyroid follicle to be monolayer growth.2. Recombinant thyroid follicle was an ideal experimental model for the research on the mechanism of regulation of thyroid function, because it both broke away from the complex environment in vivo and had integrated thyroid function. Therefore, the recombinant thyroid follicle could be used for observing the process of the production of thyroid follicle and exploring the regulatory roles of follicular structure and substances on thyroid function.3. The expressions of NIS, TG and TPO, which were all related to the synthesis of thyroid hormone, were overexpressed; the secretions of FT3 and FT4 in the supernate of culture medium were enhanced, indicating the recombinant thyroid follicle was undergoing vibrant hormonal synthesis and secretion. All evidences above proved that iodine promoted the synthesis and secretion of thyroid hormone at follicular level.4. Iodine and MMI affected the iodination of follicular TG and they had no direct effects on the expressions of TSHr and TTF-1; the CAMP/AKP and C/IP3 pathways, which belonged to TSH/TSHr signal, did not up-regulate the expressions of TTF-1 and TSHr, indicating highly iodinated TG directly resulted in the overexpressions of TTF-1 and TSHr.5. Iodine improved the iodinated extent of follicular TG and increased the expression level of TSHr through regulating TTF-1, which finally resulted in promoting the sensitivity of thyroid follicle to central TSH/TSHr signal. This is one of the important pathways for thyroid functional self-regulation.6. When TG was highly iodinated, the expressions of TSHr, NIS, TPO and TG was enhanced as well as the hormone secretion, and follicle was sensitive to central signal, which was one of the reasons for the vibrant thyroid functions.7. Different signal transduction pathways of TSH/TSHr signal played different regulatory roles on the expressions of NIS and NIS2, indicating central signals regulated the expression of different subtype of NIS at the level of mRNA splicing.In summary, we found iodine affected the iodinated extent of follicular TG from the aspect of follicle, and promoted the expression of TSHr through regulating TTF-1, which finally changed the sensitivity of follicle to central TSH/TSHr signal. TG, which was iodinated on different extent, led to the different sensitivity of follicle to central TSH/TSHr signal, this was one of the important pathways for thyroid adapting itself to different concentration of iodine and achieving functional self-regulation. The highly iodinated TG enhanced the expression of TSHr, drove follicle to be sensitive to central signal, and promoted the secretion of thyroid hormone and the expression of NIS, TPO and TG. Taken follicle as a research model, iodine efficiently expedited the synthesis and secretion of thyroid hormone through the mechanisms mentioned above.Over the past 10 years, the incidences of thyroid dysfunction, autoimmune thyroiditis and thyroid cancer that were all high iodine-related diseases obviously increased. At present little is known about how iodine regulates thyroid functions. Our work offered methodology basis and directions for the research on this area and had important theoretical and practical significance for exploring the pathogenesis of iodine-related thyroid diseases and making the scientific strategies for iodine supplement.更多还原