利用蛋白質體學方法分析甲狀腺眼病變之眼窩蛋白質變化

Analysis of the differences in protein expression associated with thyroid orbitopathy by proteomic approach

鄭凱駿(Kai-Chun Cheng)

指導教授 ： 張丞賢

高雄醫學大學/醫學院/醫學研究所/碩士(2009年)

摘要

研究目的：利用蛋白質體學的方法對甲狀腺眼病變的病人之眼外肌的肌肉組織與眼窩部的脂肪組織其蛋白質的表現作定性的分析。
研究材料及方法：本實驗從甲狀腺眼病變的病人中取得眼外肌的肌肉組織及眼窩部的脂肪組織，把這二種檢體當作實驗組，另外從接受斜視手術(手術過程需切除部分眼外肌組織)的病人中取得正常的眼外肌的肌肉組織，另外從接受眼皮整型的病人中取得正常的眼窩部的脂肪組織，把這二種檢體當作對照組，從取得的組織中去萃取蛋白質，利用二種蛋白質體的方法來鑑定實驗組與對照組之中有差異的蛋白質，第一種方法是以二維電泳膠片快速分離其中蛋白質，找出實驗組與對照組之中有差異的蛋白質點，以電噴灑離子化併四極棒飛行時間質量分析器式質譜儀鑑定出蛋白質的身份，第二種方法是以液相層析串連式質譜儀來找出實驗組與對照組之中不同的蛋白質。
研究結果：以二維電泳膠片快速分離脂肪組織中的蛋白質，比照實驗組與對照組，發現有8個蛋白質點有明顯差異，把這8個蛋白質點送去做質譜鑑定，共有4個點被鑑定出來，分別為(1)chain A, human serum albumin in a complex with myristic acid and tri-iodobenzoic acid (2)chain B, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems (3)beta globin chain variant (4)chain A, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems，這些蛋白質的濃度在實驗組中較高，有表現量增多的趨勢。為了增加鑑定的蛋白質數量，我們又採用了液相層析串連式質譜儀來比較實驗組與對照組在脂肪組織中所鑑定出的蛋白質，在實驗組中可發現有7種蛋白質是在對照組中偵測不到的，包括(1)chain A, human serum albumin in a complex with myristic acid and tri- iodobenzoic acid (2)chain B, human hemoglobin D Los Angeles: crystal structure (3)hemoglobin beta (4)chain B, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems (5)beta globin chain variant (6)chain A, cyanomet Rhb1.1 (recombinant hemoglobin) (7) unnamed protein product，在對照組中則發現有vimentin，而在實驗組中則缺乏這個蛋白質，以液相層析串連式質譜儀來比較實驗組與對照組在眼外肌的肌肉組織中所鑑定出的蛋白質，在實驗組中可發現有(1)chain A, human serum albumin in a complex with myristic acid and tri-iodobenzoic acid (2)chain B, human hemoglobin D Los Angeles: crystal structure (3) beta-globin (4)hemoglobin beta (5)chain A, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems (6)chain B, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems (7)beta globin chain variant (8)chain B, crystal structure of human hemoglobin E at 1.73 A resolution (9)unnamed protein product (10) beta globin chain，而這10個蛋白質在對照組則偵測不到。
結論：利用蛋白質體學的方法來分析，可以讓我們發現到何種蛋白質在甲狀腺眼病變中是up-regulation，何種蛋白質是down-regulation，這些資訊有助於更加瞭解甲狀腺眼病變的致病機轉，以及找尋適當的可輔助診斷的biomarkers。

關鍵字

甲狀腺眼病變 ； 蛋白質體 ； 眼外肌的肌肉組織 ； 眼窩部的脂肪組織

並列摘要

Purpose: Application of proteomics to understand the qualitative differences in protein expression of extraocular muscle tissues and orbital adipose tissues associated with thyroid orbitopathy (TO)
Material and method: We obtained extraocular muscle tissues or orbital fat tissues from patients with thyroid orbitopathy during strabismus surgery or orbital decompression as the study group. We also acquired extraocular muscle tissues from patients who received strabismus surgery or took orbital fat tissues from patients who accepted eyelid plasty as the control group. In this study, we used two proteomic methods to comprehensively examine the extraocular muscle and orbital fat proteomes of TO patients and non-TO controls. These methods included two-dimensional gel electrophoresis(2-DE) combined with electrospray ionization－quadrupole－time-of-flight mass spectrometry (ESI- QUAD-TOF mass spectrometry) and liquid chromatography tandem mass spectrometry (LC-MS/MS).
Result: Among all protein spots separated by 2DE, we found the eight spots that were significantly different between the study and the control group. The eight spots were analyzed by ESI- QUAD-TOF mass spectrometry. Proteins from four of the eight spots were identified successfully, which had relatively higher concentration in the study group. These are (1)chain A, human serum albumin in a complex with myristic acid and tri- iodobenzoic acid (2)chain B, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems (3)beta globin chain variant and (4) chain A, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems. To increase protein identification, we employed a complementary analytical platform, LC-MS/MS. By this instrument, 7 proteins were identified only in the orbital fat tissues of the study group, including (1)chain A, human serum albumin in a complex with myristic acid and tri-iodobenzoic acid (2)chain B, human hemoglobin D Los Angeles: crystal structure (3)hemoglobin beta (4)chain B, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems (5)beta globin chain variant (6)chain A, cyanomet Rhb1.1 (recombinant hemoglobin) and (7) unnamed protein product. One protein─vimentin─ was identified in the orbital fat samples of the non-TO controls and this protein had significantly expressed in the control group. There were 10 proteins identified only in the extraocular muscle tissues of the study group, including (1)chain A, human serum albumin in a complex with myristic acid and tri-iodobenzoic acid (2)chain B, human hemoglobin D Los Angeles: crystal structure (3)beta-globin (4)hemoglobin beta (5)chain A, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems (6)chain B, structure of hemoglobin in the deoxy quaternary state with ligand bound at the alpha haems (7)beta globin chain variant (8)chain B, crystal structure of human hemoglobin E at 1.73 A resolution (9)unnamed protein product (10) beta globin chain.
Conclusion: The results obtained with this proteomic analysis let us know which protein is up-regulated or down-regulated in thyroid orbitopathy. This will be of use in understanding the pathophysiology of thyroid orbitopathy as well as in finding candidates as new diagnostic biomarkers of thyroid orbitopathy.

並列關鍵字

thyroid orbitopathy ； proteomics ； extraocular muscle tissue ； orbital fat tissue

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