| Abstract: | 葛瑞夫茲氏病是一種常見的自體免疫疾病，其特徵包括甲狀腺機能亢進、眼病變及脛前黏液水腫。其中甲狀腺機能亢進約佔所有甲狀腺素機能過高 (thyrotoxicosis)原因的60 %到90 %。近年來雖然對於機能亢進疾病生理學之研究有些進展，然而對於導致此疾病的根本原因，仍瞭解甚少。本研究首先嘗試釐清葛瑞夫茲氏病病人產生非器官特異性自體抗體是否與使用抗甲狀腺藥物有關，接續探討葛瑞夫茲氏病病人甲狀腺組織增生之訊息傳遞路徑，並進而研究與甲狀腺組織增生有關之基因表達。
共140位葛瑞夫茲氏病病人參與第一部份之研究，其中32位為新診斷，54位使用propylthiouracil (PTU) 治療，54位接受methimazole (MMI)治療。病人的血清以間接免疫螢光法(indirect immunofluorescence；IIF)檢測抗嗜中性白血球細胞質抗體(antineutrophil cytoplasmic antibody；ANCA)，而用indirect passive hemagglutination 法分析抗甲狀腺微小體抗體 (anti-thyroid microsomal antibody；AMA)及抗甲狀腺球蛋白抗體 (anti-thyroglobulin antibody；ATA)；酵素聯結免疫吸收分析法（enzyme-linked immunosorbent assays; ELISA）則用以檢驗抗核抗體 (anti-nuclear antibody；ANA)、抗雙螺旋去氧核糖核酸抗體 (anti-double stranded DNA antibody；dsDNA Ab)、抗cardiolipin抗體 (IgM或IgG anti-cardiolipin antibody；IgG aCL Ab或IgM aCL Ab)、及IgG anti-β2-glycoprotein I antibody (IgGβ2GPI Ab)。TSH-binding inhibitory immunoglobulin (TBII)以radioreceptor assay檢測。結果顯示：使用MMI及PPU治療使得IgG aCL抗體產生明顯增加，但與ANA, ds DNA抗體，IgM aCL Ab, IgG β2GPI Ab並無關聯。使用MMI治療組中，ANA與ds DNA抗體呈正相關，以全部患者及MMI治療組而言，IgG aCL與IgM aCL也呈正相關。另外以PTU或MMI治療的病人中，有20.2%的病人為ANCA陽性，其絕大部份為(perinuclear-ANCA; p-ANCA) 陽性。PTU治療組中的p-ANCA陽性明顯高於MMI治療組（31.9% vs 7.1%；p=0.31）。在PTU治療組中，p-ANCA陽性的病人其平均治療時間也明顯高於p-ANCA陰性病人（32.9±16.3 vs 19.6±12.1個月；p=0.04）。這些結果顯示除了IgG aCL及p-ANCA與藥物治療有關外，其他非器官特異性抗體的產生可能是多株抗原活化（polyclonal activation）的結果。
對於葛瑞夫茲氏病甲狀腺組織增生所引發之訊息傳遞，本研究共分析38個葛瑞夫茲氏病的甲狀腺檢體，24個甲狀腺良性結節及19個正常的甲狀腺組織。由這些甲狀腺檢體所分離的蛋白質，利用西方點墨法及免疫沉澱法進行一系列訊息傳遞蛋白的表達研究，結果顯示: 1) 相較於正常甲狀腺組織，葛瑞夫茲氏病甲狀腺組織之蛋白質Raf、Erk-2及c-Jun之增加雖未達顯著，但有大於60.0% 以上的葛瑞夫茲氏病甲狀腺組織之表達量大於正常甲狀腺組織之平均值。蛋白質MEK-1則明顯增加(p<0.005)。2) PI3-K、cdc 42及Bad-p於葛瑞夫茲氏病的組織中分別約有70.0% 左右之檢體比正常甲狀腺組織表現之平均值增加； Akt-p及cdk2-cyclin E則明顯上升(p<0.001)。3) 有55.3%的葛瑞夫茲氏病甲狀腺組織其Rb-E2f之表達比正常甲狀腺組織少，但Rb-p 的表現量則有63.2%的葛瑞夫茲氏病甲狀腺組織增加。4) P38-p、IAP及caspase 3 之表現在葛瑞夫茲氏病、甲狀腺結節及正常甲狀腺組織中並無明顯差異。5) 有高達90.0% 以上的葛瑞夫茲氏病甲狀腺組織其之HGF之表達量大於正常甲狀腺組織之平均值；而Met及TSH-R於葛瑞夫茲氏病之甲狀腺組織表現增加(p<0.05)。由此可知，葛瑞夫茲氏病甲狀腺增生之訊息傳遞主要經由兩條signal pathways: Raf/MEK-1/Erk-2使細胞增生以及PI3-K/Akt/Bad-p或PI3-K/Cdc42/Bad-使細胞存活。
而於甲狀腺組織增生有關之基因表達方面，我們以RT-PCR半定量方法探討血管生成因子如內皮素(endothelin 1 [ET-1]) 及其受體 ETa、ETb，血管內皮生長因子(vascular endothelial growth factor [VEGF]) 及其受體flt-1、KDR，及細胞生長因子如類胰島生長因子-1 (insulin-like growth factors [IGF-1]) 及其受體 IGF-1R等之mRNA表達量。共分析5個正常甲狀腺組織，5個甲狀腺乳突癌及14個葛瑞夫茲氏病之甲狀腺組織，其結果如下：1) 在血管內皮生長因子 (VEGF) 基因方面，與正常組織比較，葛瑞夫茲氏病之VEGF、flt-1與KDR明顯增加（p<0.005）。2)在內皮素 (ET-1) 基因方面，葛瑞夫茲氏病（p<0.05）及乳突癌（p<0.005）的表現皆增加。ETa於葛瑞夫茲氏病之表現減少（p<0.05）而ETb於乳突癌之表現減少（p<0.001）。3)在類胰島生長因子-1 (IGF-1)基因表現方面，葛瑞夫茲氏病之表達量增加（p<0.05），但於IGF-1R則不見增加。
本實驗的發現將有助於瞭解葛瑞夫茲氏病甲狀腺增生之訊息傳遞路徑，同時幫助釐清甲狀腺增生之分子作用機轉，亦有助於新藥的開發，或可因而免除病人反覆發病及開刀之苦。
Graves’ disease is a potentially severe autoimmune disease, characterized by combinations of either hyperthyroidism, ophthalmopathy, or pretibial myxedema. Graves’ hyperthyroidism accounts for 60 % to 90 % of thyrotoxicosis. Though there is progress in understanding of pathophysiology for Graves’ disease, little is known about its etiology. In the present study, we investigated the relationship between non-organ specific autoantibody production relating to medical treatment, and also on the signal transduction pathways and gene expressions of thyroid hyperplasia in patients with Graves’ disease.
One hundred and forty patients with Graves’ disease (32 new patients, 54 treated with propylthiouracil (PTU) and 54 treated with methimazole (MMI) were included in the first study. Sera were screened for antineutrophil cytoplasmic antibody (ANCA) by indirect immunofluorescence (IIF). Thyroid autoantibodies against microsomal antibodies (AMA) and thyroglobulin antibodies (ATA) were detected by indirect passive hemagglutination assays, while thyroid autoantibodies against thyrotropin receptor (TRAbs) by a radioreceptor assay. The other non-organ-specific autoantibodies, i.e., anti-nuclear antibody (ANA), anti-double stranded DNA antibody (dsDNA Ab), IgM or IgG anti-cardiolipin antibody (IgM or IgG aCL Ab), and IgG anti-β2-glycoprotein I antibody (IgG β2GPI Ab) were tested by ELISA. The results showed that treatment with MMI or PTU produced a significant difference in IgG aCL Ab production but not in ANA, dsDNA Ab, IgM aCL or IgG β2GPI. For those treated with MMI but not those treated with PTU, ANA and anti-dsDNA Ab were positively correlated. IgG and IgM aCL Ab were positively correlated overall and for those on MMI but not PTU treatment. There were 20.2 % of patients with Graves’ disease receiving PTU and MMI, who were seropositive for ANCA. All of them were p-ANCA positive. The frequency of p-ANCA positive status in the PTU treatment group was significantly higher than in the MMI treatment group (31.9% vs. 7.1%; p=0.01). In the PTU treatment group, the average duration of treatment in p-ANCA positive patients was significantly longer than in p-ANCA negative patients (32.9±16.3 vs. 19.6±12.1 months, p=0.04). These results indicated that treatment with MMI or PTU produced a significant difference in IgG aCL Ab and p-ANCA production and that the appearance of the non-organ-specific autoantibodies is probably largely a coincidental effect of polyclonal activation.
As for signaling pathways of thyroid hyperplasia in Graves’ disease, we investigate the protein expressions by using western blot and immunoprecipitation of different kinase in 38 Graves’ thyroid tissues, 24 benign thyroid nodules and 19 normal thyroid tissues. The results showed that: 1) Compared to those of normal thyroid tissues, the expression amounts of Raf, Erk-2 and c-Jun show no significanct difference. But there are more than 60.0% of the Graves’ thyroids exceed the average of normal thyroid tissue in these three protein expression. The MEK-1 expression is increased significantly in the Graves’ thyroids (p<0.005). 2) The expression amounts of PI3-K, cdc42 and Bad-p show no significant difference among the three groups, but there are still more than 70.0% of the Graves’ thyroids exceed the average of normal thyroid tissue in these three protein expression. The expression of Akt-p and cdk2-cyclin E is increased significantly in the Graves’ thyroids (p<0.001). 3) For Rb-E2f, 55.3% of the Graves’ thyroids express less than the average of normal thyroid tissue, while there are 63.2% of the Graves’ thyroid increased in Rb-p expression. 4) There is no significant difference between the expression amounts of P38-p, IAP and caspase3 among the three groups. 5) More than 90.0% of the Graves’ thyroids express more HGF than the average of normal thyroid tissue. The expression amounts of Met and TSH-R are significantlt increased in the Graves’ thyroids (p<0.05). Hence, two major signal pathways are newly disclosed to be involved in the thyroid hyperplasia in Graves’ disease: through Raf/MEK-1/Erk-2 for cell proliferation; through PI3-K/Akt/Bad-p or PI3-K/Cdc42/Bad-p for cell survival.
For thyroid gene study, semiquantitative RT-PCR was used to determine the mRNA expression of some vasoregulatory mediators, such as endothelin 1 (ET-1) and its receptors ETa, ETb; vascular endothelial growth factor (VEGF) and its receptors flt-1, KDR; insulin-like growth factors (IGF-1) and its receptors IGF-1R. Five normal thyroid tissues, 5 thyroid papillary carcinoma (PTC) and 14 Graves’ thyroid specimen were analyzed. The results showed that: 1) the expressions of VEGF, flt-1 and KDR were significantly increased in Graves’ disease (p<0.005); 2) the expression of ET-1 gene was increased significantly in both PTC patients (p<0.005) and Graves’ patients (p<0.05). ETa expression was decreased in Graves’ patients (p<0.05), while ETb was decreased in PTC patients (p<0.001); 3) the expression of IGF-1, but not IGF-1R gene, showed increase in Graves’ patients (p<0.05).
The dissection of the molecular mechanism in our findings will clarify the pathogenesis of Graves’ disease. We believe that the realization of disease entity might give a new insight into novel therapeutic development, and finally to prevent disease relapse and thyroid surgery. |
