莊立民臺灣大學:臨床醫學研究所陳美秀Chen, Mei-HsiuMei-HsiuChen2010-05-102018-07-062010-05-102018-07-062008U0001-3007200813302000http://ntur.lib.ntu.edu.tw//handle/246246/181641葛瑞夫茲氏眼病變(Graves’ ophthalmopathy, GO)，或稱為甲狀腺眼病變(thyroid eye diease, TED)。 病人會有凸眼(proptosis)、眼瞼內縮(lid retraction)、眼瞼水腫(lid edema)，眼外肌(extraocular muscles)病變引起複視(diplopia)等影響美觀、視力的症狀。凸眼的原因包括眼外肌及眼窩脂肪組織的肥大。 而脂肪組織的病變，在最近10年對於脂肪組織有完全改觀的認知之後，更顯得有進一步研究的必要。 由於凸眼的治療目前僅有外科手術一途，我們也希望研究凸眼患者眼窩脂肪組織肥大的分子機轉能幫助找出內科治療或者預防發生的可能性。 相對於葛瑞夫茲氏病中甲狀腺功能亢進的致病機轉的了解，葛瑞夫茲氏眼病變的致病機轉較不是那麼清楚。 由於葛瑞夫茲氏病是一種自體免疫疾病，相關的發炎反應引起很多cytokines (或稱chemokines)的釋放，早期的研究偏重此方面的研究，而且是偏重淋巴球及纖維母細胞釋放cytokines的研究。 近幾年，發現了脂肪細胞也會分泌adipokines，除了影響代謝，也與發炎反應有關。脂肪組織中也發現形態上類似纖維母細胞，但是行為上是偏向於脂肪細胞的脂肪前驅細胞(preadipocyte)。 在受到刺激後脂肪前驅細胞可以分化成脂肪細胞，這樣的過程稱之為adipogenesis。 前幾年的眼窩脂肪組織研究重心在adipogenesis，主要使用isolated orbital fibroblasts來做細胞培養分化的研究。 我們認為脂肪組織包含不只脂肪細胞，必須要有全組織的研究才能有全面的了解。 運用cDNA微陣列(cDNA microarray)建立疾病假說是不錯的選擇。但是由於基因為數過大，而且個人的差異又很大，再加上人體組織的檢體通常數量很少，以致於難以區分分析結果到底是真的有不同還是干擾。 所以，我們使用基因群組增強分析(gene sets enrichment analysis, GSEA)，它是依生理途徑(biological pathway)作為分群的依據，所得結果較具生理的意義。 最後，進一步用定量即時聚合酵素連鎖反應(quantitative real-time PCR, QPCR)來做單一基因的測試，以證實基因的參與。結果發現，溶酶體相關基因群組與眼窩脂肪組織肥大有關。 其中，ceroid-lipofuscinosis, neuronal 2, late infantile (CLN2)，ceroid-lipofuscinosis, neuronal 3, juvenile (CLN3)以及β subunit of hexosaminidase A (HEXB) mRNA表現的減少與眼窩脂肪組織肥大有關。 此外，我們注意到代表巨噬細胞標誌的CD68基因也在溶酶體相關基因群組中，所以我們也針對巨噬細胞的浸潤情形及原因，做了相關的研究。 研究發現巨噬細胞在葛瑞夫茲氏眼病變病人眼窩脂肪組織的浸潤增加，而這種現象與肥胖個體脂肪組織的巨噬細胞的浸潤一樣與C-C motif chemokine family ligand 2 (CCL2)，也稱之為monocyte chemoattractant protein-1 (MCP-1)基因有關。 雖然，我們目前的研究結果尚不足以找出治療或預防眼窩脂肪組織肥大的方式，卻提供了整體可行的研究方向。Graves’ ophthalmopathy (GO) or thyroid eye disease (TED) is associated with manifestations of proptosis, lid retraction, lid edema and extraocular muscles restriction related diplopia, which may influence patients’ cosmetics and visual acuity. Causes of proptosis included hypertrophy of extraocular muscle and orbital fat。 We are interested in orbital fat pathology as we know more about the new conepts of adipose tissue in its role in encocrine and inflammatory physiology as well as adipogensis in the recent 10 years. The only treatment for proptosis now is surgery. We hope the study about the molecular mechanism of Graves’ ophthalmoapthy will help to find out the possible medical treatment or the way to prevent it. In contrast to the understanding of the patholgenesis of hyperthyroidism in Graves’ disease, the pathogeneis of Graves’ ophthalmopathy is not so clear. Since Graves’ disease is an autoimmune disease, earlier studies focused in the release of cytokines (or chemokines) from lymphocytes and fibroblasts. However, in recent 10 years, we know that adipocytes could secret adipokines which will influence metabolism and inflammatory process and the preadipocytes which reside in adipose tissue have the ability to differentiate to adipocyte. After knowing that, studies began with adipogenesis in orbital fat using isolated orbital fibroblasts. We know that adipose tissue is a heterogenous tissue with multiple cell types, the studies using isolate orbital fibroblast would see only part of the mecahnism. For us to understand the whole tissue pathology, we need cDNA microassay to generate the hypothesis about the molecular mechanism of orbital fat hypertrophy. However, the large number of genes tested, the high variability between individuals, and the limited samples sizes in human studies have made it difficult to distinguish true differences from noise. The Gene Sets Enrichment Analysis (GSEA) is a powerful method to avoid the noise. By using the GSEA method, we found the expressions of specific gene sets related to lytic vacuoles, lysosomes and vacuoles, which we termed lysosome-related genes, were substantially different between orbital adipose tissues obtained from patients with and without Graves’ ophthalmopathy. We also use real-time quantitative PCR (QPCR) to validate single gene under those gene sets, which revealed the expression of CLN2, CLN3, and HEXB were down-regulated in orbital fat of patients with Graves’ ophthalmopathy. We also noticed that the macrophage marker, CD68, is a member of the lysosome-related gene set. We tried to elucidate the infiltration macrophage in orbital fat and its associated mechanism. We demonstrated macrophage infiltration is increased in orbital fat of patients with Graves’ ophthalmopathy and the mechanism is similar to the mechanism of macrophage infiltration in adipose tissue of obese subjects, to be associated with C-C motif chemokine family 2 (CCL2), or monocyte chemoattratant protein-1 (MCP-1). We understood that what we have done is not sufficient for generating medical treatment or way to prevent Graves’ ophthalmopathy, but our work provided a workable way to it.口試委員會審定書……………………………………………………………………...i謝………………………………………………………………………………..…….ii文摘要………………………………………………………………………………..iii文摘要………………………………………………………………………………..v錄……………………………………………………………………………………viii目錄…………………………………………………………………………………xiii目錄…………………………………………………………………………………xiv一章 緒論………………………………………………………………………….....11.01葛瑞夫茲氏病(Graves’ disease)的臨床表現…………………………………1.011甲狀腺功能亢進的臨床症狀……………………………………………1.012甲狀腺及甲狀腺外的表徵………………………………………………1.02葛瑞夫茲氏病中甲狀腺功能亢進的致病機轉………………………………2.03葛瑞夫茲氏病引起甲狀腺功能亢進的治療………………………………..3.031藥物治療………………………………………………………………..3.032放射線治療……………………………………………………………..4.033外科手術治療…………………………………………………………..4.04葛瑞夫茲氏眼病變(Graves’ ophthalmopathy)的臨床表現…………………5.05 影像學上所觀察到的眼病變型態…………………………………………6.06葛瑞夫茲氏眼病變的組織病理…………………………………………….6 1.061眼外肌(extraocular muscles)………………………………………….71.062眼窩脂肪組織(orbital adipose tissue)………………………………...7.063苗勒氏肌(Müller''s muscle)…………………………………………...8.07 葛瑞夫茲氏眼病變與Adiposity……………………………………………8.08葛瑞夫茲氏眼病變的治療…………………………………………………..9.081腎上腺皮質類固醇(adrenal corticosteroids)…………………………..9.082 Octreotide………………………………………………………………9.083放射治療.……………………………………………………………….10.084外科手術.……………………………………………………………….10.09葛瑞夫茲氏眼病變的致病機轉研究近況………….……………………….11.091自體抗原(autoantigen)及動物模式……………………………………12.092眼窩纖維母細胞(orbital fibroblast)及脂肪增生(adipogenesis)……….12.093 黏多醣(glycosaminoglycan)堆積……………………………………..14.094遺傳及環境的影響…………………………………………………….15.095 細胞激素(cytokines)…………………………………………………..15.10眼窩脂肪組織肥大病因研究之重要性、研究假說及研究的目的………..16.101眼窩脂肪組織的異質性(heterogenesity)……………………………..17.102基因群組增強分析(gene sets enrichment analysis)…………………..18.103假說的建立及研究目的………………………………………………19二章 研究方法與材料……………………………………………………………...20.1 人類眼窩脂肪組織的取得…………………………………………………..20.2 RNA的分離………………………………………………………………...20.3 基因微陣列檢體(microarray)的準備，雜交(hybridization)，掃描審視(scanning)…………………………………………………………………………21.4 微陣列資料正常化(microarray data normalization)及分析………………...21.5定量即時聚合酵素連鎖反應 (quantitative real-time PCR)…………………22.6免疫組織螢光分析( immunohistochemistry)………………………………...23.7 統計分析……………………………………………………………………..24三章 結果 ……………………………………………………………………….25.1葛瑞夫茲氏眼病變病人眼窩脂肪組織肥大與溶酶體相關基因有關………25.2基因篩選………………………………………………………………………25.3 葛瑞夫茲氏眼病變病人眼窩脂肪組織肥大與CLN2, CLN3, HEXB基因表現減少有關……………………………………………………………………….26.4使用CLN2抗體的免疫組織螢光染色主要在非脂肪細胞部分……………27.5 葛瑞夫茲氏眼病變病人眼窩脂肪組織肥大與CD68 gene 表現增加有關..27.6 巨噬細胞在葛瑞夫茲氏眼病變病人眼窩脂肪組織的浸潤增加…………..27.61 巨噬細胞在葛瑞夫茲氏眼病變病人眼窩脂肪組織中分佈的情形…27.62 巨噬細胞浸潤的程度…………………………………………………28.63 巨噬細胞浸潤在病程較長的病人眼窩組織中也可見到……………28.7巨噬細胞在葛瑞夫茲氏眼病變病人眼窩脂肪組織的浸潤增加與CCL2 (MCP-1)基因有關………………………………………………………………...28四章 討論…………………………………………………………………………...30.1基因群組增強分析的微陣列研究給我們更具有生理意義的疾病假說…...30.2溶酶體…………………………………………………………………………32.21溶酶體分布在不同細胞有不同名稱…………………………………..33.22溶酶體相關基因在葛瑞夫茲氏眼病變病人眼窩脂肪組織中可能扮演的角色……………………………………………………………………….34.221 CLN2及CLN3…………………………………………………35.222 HEXB……………………………………………………………36.223 CD68……………………………………………………………..37.3 巨噬細胞在葛瑞夫茲氏眼病變眼窩脂肪組織病理中所扮演的角色……..38.4眼窩脂肪組織中各種細胞及基因的變化與脂肪組織肥大的關係…………42.5脂肪組織具有多元的生理及病理角色以及區域特異性，葛瑞夫茲氏眼病變眼窩脂肪組織肥大也是其中的一部分………………………………………….42.51脂肪組織是內分泌器官……………………………………………….43.52脂肪組織是發炎器官………………………………………………….44.53脂肪組織的區域特異性……………………………………………….45五章 展望…………………………………………………………………………...46.1研究結果在臨床上的應用及貢獻……………………………………………46.11從了解病人的需要及治療的困難處著手……………………………..46.12繼往開來，全面檢討…………………………………………….…….46.13確實可行的方向………………………………………………………..47.2研究工作於將來的研究方向及貢獻…………………………………………48.21基礎方面………………………………………………………………..48.211眼病變中的脂肪組織病理………………………………………48.2111脂肪細胞脂質堆積………………………………………..49.2112纖維母細胞分泌及降解glycosaminoglycans的機轉……49.2113 各種細胞內溶酶體與antigen presenting的關係………..49.2114脂肪細胞、纖維母細胞，以及巨噬細胞的共同培養……..50.212脂肪組織病理研究的擴大………………………………………50.22臨床方面………………………………………………………………..51.3結語……………………………………………………………………………52六章 論文英文簡述………………………………………………………………...53考文獻……………………………………………………………………………….67錄…………………………………………………………………………………….81目錄1. 由上而下：眼瞼內縮(lid retraction)、眼瞼水腫(lid edema)、凸眼(proptosis)…...812. 凸眼側面觀………………………………………………………………………823. orbital CT, coronal(上) and sagittal(下) view：眼窩脂肪組織肥大合併眼外肌肥大………………………………………………………………………….……………834. orbital CT, coronal(上) and sagittal(下) view：只有眼窩脂肪組織肥大………..845. orbital CT, coronal(上) and sagittal(下) view：只有眼外肌肥大……….……….856. 脂肪細胞分化不同的時程………………………………………………………867. 脂肪細胞分化，一些原本抑制分化的基因表現減弱…………………………..878. 定量即時聚合酵素連鎖反應結果………………………………………………889. 脂肪組織冷凍切片使用anti-CLN2抗體的免疫組織化學染色…….…………8910. 葛瑞夫茲氏眼病變眼窩脂肪組織肥大與CD68 表現增加有關………….….9011. 免疫組織染色觀察巨噬細胞在眼窩脂肪組織中的浸潤……………………..9112. 巨噬細胞浸潤的程度…………………………………………….…………….9213. 葛瑞夫茲氏眼病變眼窩脂肪組織中，巨噬細胞浸潤的程度增加………....9314. 巨噬細胞浸潤為一種慢性的過程……………………………………………9415. 葛瑞夫茲氏眼病變眼窩脂肪組織的CCL2 (MCP-1) mRNA表現增加……9516. 巨噬細胞在葛瑞夫茲氏眼病變眼窩脂肪組織的浸潤增加與CCL2 (MCP-1) 基因有關…………………………………………………9617. Oil-Red染色於3T3-L1脂肪細胞分化初期…………………………………..9718. 眼窩脂肪組織中各種細胞及基因的變化與脂肪組織肥大的關係…………98目錄1. Demographics and clinical characteristics of patients with Graves’ ophthalmopathy and controls………………………………1002. Primers for QPCR of candidate genes, CD68, CCL2 (MCP-1), osteopontin and β-actin………………………………………………1013. Summary results of GSEA of the mRNA expressions between subjects with Graves’ ophthalmopathy v.s. controls………………………………………………1024. Details of the selected individual genes in gene ontology ID 323, 5764, 5773………v1035. Characteristics of lysosome and related organelles……………………………104application/pdf1155598 bytesapplication/pdfen-US葛瑞夫茲氏眼病變脂肪組織微陣列基因群組增強分析溶酶體巨噬細胞Graves'' ophthalmopathyadipose tissuemicroarrayGene Sets Enrichemnt Analysis (GSEA)lysosomemacrophage[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/181641/1/ntu-97-D89421009-1.pdf