Study on the Pathogenesis of Graves' Ophthalmopathy using 3T3-L1 Cells Which Express Thyrotropin Receptor

甲状腺素受体（TSHR）可能引起Graves'眼病患者眶后组织的自身免疫反应。结果：TSHR基因在胰岛素、地塞米松和IBMX存在下表达，引物延伸和克隆的cDNA序列显示TSHR基因在大鼠脂肪组织中的转录存在于-89 ~-68之间的多个位点。TSH依赖性下调TSHR观察后1小时，TSH添加一个不需要蛋白质合成。下调是cAMP依赖性的。DNase I保护实验表明，分化后的3 T3-L1细胞核提取物对含有cAMP反应元件的序列-146 ~-127具有保护作用。此外，我们发现了-127的下游另一个保护位点。用针对许多已知转录因子的抗体进行的凝胶位移分析显示，与下游元件结合的转录因子对脂肪细胞是特异性的。我们现在正在鉴定与下游元件结合的分子。最后，我们研究了不同的PPAR<gamma>_ 2激活剂和重组腺病毒对3 <gamma><gamma>T3-L1细胞的作用，发现PPAR_ 2激活剂和重组腺病毒能促进3 T3-L1细胞的分化和TSHR的表达。TSHR的表达伴随着TSH刺激的cAMP生成。内容提要：脂肪细胞的分化与TSHR mRNA的表达密切相关，TSHR在脂肪细胞中的大量表达有着复杂的机制。

It has been speculated that thyrotripin Receptor (TSHR) elicites the autoimmune reaction in retroorbital tissues in Graves' patlents and causes the ophthalmopathy. We investigated the molecular mechanisms of expression in rat adipocytes and 3T3-L1 cells.Results : TSHR is expressed in the presence of insulin, dexamethazone, and IBMX.Primer extension and cloned cDNA sequences showed that transcription of TSHR gene in rat adipose tissue was from multiple stasites between -89 to -68. TSH-dependent down regulation of TSHR was observed 1 hr after TSH addition a dis not need protein synthesis. Down regulation was cAMP-dependent. DNase Iprotection assay showed 1 nuclear extracts from differentiated 3T3-L1 cells protected sesquence-146 to -127 which contained cAMP-responsive element. Furthermore, we found another protection site downstream of-127. Gel shift analysis with antibodies against many known transcription factors revealed that the transcription factor (s) which bound to the downstream elements is (were) secific to the adipocytes. We are now identifying the molecule which binds to the downstream element. Finally, we evaluated the effects of various PPAR_<gamma> activators and recombinant adenovirus which expresses PPAR_<gamma>2, and we found that PPAR_<gamma> activators and recombinant adenovirus stimulated the differentiation and the expression of TSHR in 3T3-L1 cells. Express of TSHR was accompanied by the TSH-stimulated cAMP prodction.Summary : Differentiation of adipocytes are closely related to the expression of TSHR mRNA.There is a complicated mechanism which allow the a enormous amount of expression of TSHR in adipocytes.