臺灣博碩士論文加值系統

成人發作型史笛兒氏症 (Adult-onset Still’s disease, AOSD) 為不明原因發燒之自體發炎疾病，並有文獻指出 AOSD 在亞洲地區的死亡率較高。全身性紅斑性狼瘡 (Systemic Lupus Erythematosus, SLE) 為慢性自體免疫疾病，起因於自體抗體攻擊各器官而導致的全身性疾病。目前 AOSD 及 SLE 的確切致病機制仍不明確，但是這兩種疾病的部分症狀與治療方式類似。蛋白質體學 (Proteomics) 隨著質譜儀的迅速發展，被廣泛應用於各種研究領域。非標定 (label-free) 質譜定量技術為近年來廣泛採用於蛋白質體學的質譜定量方式之一，而 Sequential window acquisition of all theoretical fragment ion spectra (SWATH) 是目前新穎之非標定質譜定量技術。因此本實驗將利用蛋白質體學的角度搭配 LC-MS/MS，探討 AOSD 與 SLE 疾病對人體蛋白質的影響，並從中找出具有表現差異之蛋白進行分析，實驗之結果證實 AOSD 與 SLE 之間確實有一定程度之相關性，並且兩種疾病皆有免疫系統與甲狀腺異常之情形發生，但是 AOSD 與 SLE 之免疫系統異常分別主要為補體系統異常與凝血系統異常；AOSD 之甲狀腺異常主要為細胞激素分泌異常造成甲狀腺功能障礙，而 SLE 之甲狀腺異常則因為免疫系統攻擊甲狀腺所導致，其餘脂質相關與急性蛋白異常路徑也皆與兩種疾病的致病機制具相關性。

Adult-onset Still’s disease (AOSD) is a systemic multisystemic disease of unknown origin that usually occurred in young adults. Systemic Lupus Erythematosus (SLE) is an autoimmune disease, characterized by discoid rash, malar rash and nonerosive arthritis. Because AOSD and SLE share similarities in the clinical manifestations, proteomic profiles in AOSD, SLE and normal specimens were compared using label-free quantitative proteomic approach. Sequential Window Acquisition of all THeoretical fragment ion spectra (SWATH) was the most innovative label-free quantitative technology and was used in this study. Therefore, we use ultra performance liquid chromatography (UPLC) coupled with Q-TOF MS, and SWATH technology is used to investigate the pathological mechanism of AOSD and SLE protein expression changes and to find relevant pathways and biomarkers respectively. In our results, AOSD and SLE was shared some degree of association in pathological mechanism. Immune system and thyroid abnormalities occurred in the two diseases simultaneously. However, the immune system abnormalities in AOSD and SLE were resulted from complement and coagulation system aberrant, respectively. The thyroid abnormalities in AOSD mainly due to abnormal secretion of cytokines caused by thyroid dysfunction. The thyroid abnormalities in SLE are caused by the immune system attack thyroid. The other lipid-related and acute protein abnormalities were also associated with the pathogenesis of two diseases.

中文摘要 i
Abstract ii
英文縮寫對照表 iii
第一章 緒論 1
第一節 前言 1
第二節 成人發作型史笛兒氏症簡介 2
2-1 成人發作型史笛兒氏症的臨床症狀 2
2-2 成人發作型史笛兒氏症的病因和病理機制 2
2-3 成人發作型史笛兒氏症的病程及診斷 7
2-4 成人發作型史笛兒氏症的治療 8
第三節 全身性紅斑性狼瘡簡介 9
3-1 成人發作型史笛兒氏症的臨床症狀 9
3-2 全身性紅斑性狼瘡的病因和病理機制 10
3-3 全身性紅斑性狼瘡的診斷 14
3-4 全身性紅斑性狼瘡的治療 14
3-5 全身性紅斑性狼瘡於質譜上的研究 15
第四節 蛋白質體學 16
4-1 膠體分析方式 17
4-2 層析分析方式 18
4-3 標定質譜定量技術 19
4-4 非標定質譜定量技術 20
第五節 研究動機與目的 23
第六節 實驗流程 24
第二章 研究材料與方法 25
第一節、血清樣品收集 25
第二節、血清蛋白質萃取及定量 28
2-1 人類血清蛋白質萃取 28
2-2 人類血清蛋白質定量 29
第三節、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 29
3-1 11% SDS-PAGE 29
3-2 28% SDS-PAGE 30
第四節、銀染 (silver stain) 31
第五節、In tube digestion 之膠體製作 31
第六節、膠體內水解 (In gel digestion) 32
第七節、Zip Tip 去鹽純化 33
第八節、液相層析串聯式質譜儀 33
8-1 液相層析-串聯式質譜儀 (LC-MS/MS) 33
8-2 層析梯度條件最佳化測試 34
8-3 標準品測試 36
8-4 真實樣品之人類血清蛋白上機 37
第九節、 以 Protein PilotTM Software 進行蛋白質身分鑑定 39
第十一節、蛋白質功能性分析 (Gene Ontology analysis, GO analysis) 42
第十二節、 KEGG 路徑 42
第三章 結果 43
第一節 血清蛋白質萃取方法可行性確認 43
第二節、11% SDS-PAGE 44
第三節 28% SDS-PAGE 44
第四節 液相層析串聯式質譜分析 (LC-MS/MS) 44
4-1 層析梯度條件最佳化測試 44
4-2 內標準品濃度測試 45
4-3 真實樣品上機 46
第四章 討論 49
4-1 成人發作型史笛兒氏症之探討 49
4-2 全身性紅斑性狼瘡之探討 53
第五章 結論 57
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