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研究生:周涵雯
研究生(外文):Han-Wen Chou
論文名稱:血清中新穎性抗4-羥基-2-壬烯醛修飾α1-抗胰蛋白酶胜肽自體抗體同型於台灣女性修格蘭氏症病人的探討
論文名稱(外文):The investigation of autoantibody isotypes against novel 4-hydroxy- 2-nonenal-modified α1-antitypsin peptide adduct in serum from Taiwanese females with Sjögren''s syndrome
指導教授:林景堉
指導教授(外文):Ching-Yu Lin
口試委員:陳威戎張棋楨鄭朝文
口試委員(外文):Wei-Jung ChenChi-Ching ChangChao-Wen Cheng
口試日期:2017-07-18
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:57
中文關鍵詞:原發性修格蘭氏症4-羥基-2-壬烯醛
外文關鍵詞:Sjö4-Hydroxy-Trans-2-NonenalHNEalpha-1-antitrypsinAlpha-1-acid glycoprotein 1
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修格蘭氏症候群(Sjögren''s syndrome)一般俗稱乾燥症候群,它是一種病因尚未完全明瞭的外分泌腺自體免疫疾病[12,15]。外分泌腺受到淋巴球浸潤後長期引起發炎,最後導致乾口症,乾燥性角膜結膜炎與唾液腺腫大;少數病人會併發腺體外自體免疫症狀。由於修格蘭氏症候群病程相當緩慢從發病到確診平均約五到十年,我們本次鎖定研究主軸為4-羥基-2-壬烯醛(4-Hydroxy-Trans-2-Nonenal, HNE)在修格蘭氏症候群致病機制的角色。在流行病學統計中,修格蘭氏症的風險包含年齡、性別、種族,在血清學檢測包含Anti-SSA(Ro)和Anti-SSB(La)。
本次研究使用98例女性血清樣本(49例原發性修格蘭氏症與49例年齡配對健康控制組)。我們挑選9對檢體探討血清中特定抗體,其檢體須經去白蛋白和去免疫球蛋白G、一維凝膠電泳和奈米級液相層析串聯質譜LC-MS/MS從血清樣品中篩選候選的4-羥基-2-壬烯醛-修飾胜肽片段。
將80例檢體,以西方墨點法和免疫沉澱法檢驗原發性修格蘭氏症血清與健康控制組中4-羥基-2-壬烯醛-蛋白質加成物測蛋白質量。再用免疫酵素連結反應(ELISA)檢測49例健康對照組、49例原發性修格蘭氏症患者、31例紅斑性狼瘡患者、40例類風濕性關節炎患者之血清4-羥基-2-壬烯醛-胜肽加成物量及自體抗體同型分析。經由LC-MS/MS分析和西方墨點法鑑定出原發性修格蘭氏症和健康對照組中,血清蛋白差異2倍以上的有兩個分別為alpha-1-antitrypsin (A1AT)和Alpha-1-acid glycoprotein 1(AGP)。在血清蛋白質的表現量上,這兩個4-羥基-2-壬烯醛-胜肽加成物量在原發性修格蘭氏症和健康控制組沒有差異。評估A1AT胜肽是原發性修格蘭氏症患者血液的自體抗原,未修釋胜肽 (A1AT 50-63) 與4-羥基-2-壬烯醛修飾胜肽 (A1AT 50-63) 能夠刺激原發性修格蘭氏症患者產生自體抗體且與健康受試者比較具統計顯著。本次研究目的,確立血清中4-羥基-2-壬烯醛含量、4-羥基-2-壬烯醛修飾胜肽等研究外,整體研究方向是以蛋白質體學的方式進行,這次研究血清檢體去蛋白以一維的SDS-PAGE分離,在膠體內分解處理後進入奈米級液相層析串聯質譜中分析、量化,相對定量於原發性修格蘭氏症患者和健康受試者檢體血清中修飾與未修飾之4-羥基-2-壬烯醛胜肽,依據此模式繼續尋找血清中新穎的生物標記。將本次研究的發現,我們會評估所鑑定出4-羥基-2-壬烯醛胜肽加成物是否在自體抗體同型中作為原發性修格蘭氏症的診斷生物標記。
Sjögren''s syndrome is commonly known as dry syndrome, it is an etiology is not yet fully understood exocrine autoimmune disease [12,15]. Exocrine glands by lymphatic infiltration caused by long-term inflammation, and finally lead to dry mouth disease, dry keratoconjunctivitis and salivary gland enlargement; a small number of patients with concurrent glandular autoimmune symptoms. Due to the relatively slow course of the repair of the disease, the average duration of the study is about 5 to 10 years from the onset to the diagnosis. We are now targeting the 4-hydroxy-2-nonenal (HNE) The role of the pathogenesis of the disease. In epidemiological statistics, the risk of repairing streptosis includes age, sex, and race, including anti-SSA (Ro) and anti-SSB (La) in serological tests.
In this study, 98 cases of female serum samples (49 cases of primary Sjögren''s syndrome and 49 cases of age matched health control group). We selected 9 pairs of samples to explore specific antibodies in serum, the subject to de-albumin and de-immunoglobulin G, one-dimensional gel electrophoresis and nano-liquid chromatography tandem mass spectrometry LC-MS / MS from the serum samples The candidate 4-hydroxy-2-nonenal-modified peptide fragment was screened.
The protein content of 4-hydroxy-2-nonenal-protein adduct in primery Sjögren''s syndrome patiens serum and healthy controls by western blotting and immunoprecipitation. This study 49 cases of healthy controls, 49 patients with primary Sjögren''s syndrome, 31 patients with lupus erythematosus and 40 patients with rheumatoid arthritis were treated with immunolytic enzyme-linked immunosorbent assay (ELISA). LC-MS / MS analysis and western blot method, there were two positive differences between the alpha-1-antitrypsin (A1AT) and the alpha -1-acid glycoprotein 1 (AGP). There was no difference in the HNE-modifed A1AT peptide between the primery Sjögren''s syndrome and healthy controls. Can stimulate the primary Sjögren''s syndrome of patients with streak disease to produce autoantibodies and compared with healthy subjects statistically significant. The purpose of this study is to establish the serum 4-HNE, 4-HNE aldehyde modified peptides and other studies, the overall research direction is to protein-based way.These proteins weer detected by one-dimensional SDS-PAGE and analyzed in the colloid decomposition process. The samples were analyze and quantified in the nanometer liquid chromatography-tandem mass spectrometry, and the patients were treate primery Sjögren''s syndrome.
目錄
第一章、緒論 15
一、 原發性修格蘭氏症流行病學 15
二、原發性修格蘭氏症候群診斷標準 17
三、原發性修格蘭氏症候診斷方式 18
四、氧化壓力 19
五、脂肪過氧化 - 4-羥基壬烯醛(4-hydroxy-2-nonenal , 4-HNE) 20
六、Alpha-1 antitrypsin(α-1抗胰蛋白酶) 22
七、Alpha-1 acidglycoprotein(α-1-酸性糖蛋白) 22
八、研究動機 23
第二章、研究策略 25
第三章、研究方法與材料 26
一、樣品蒐集與製備 26
二、質譜分析 26
2-1、膠體內水解 ( In-Gel digestion ) 27
2.3蛋白質的溶液內水解(Protein digestion in-solution digestion ) 28
2-4、經奈米級液相層析串聯質譜 ( nano-LC/MS/MS ) 4-羥基反式2-壬烯胜肽的鑑定 28
2-5、資料庫檢索 29
三、西方墨點法(Western blot) 30
四、免疫沉澱法(Immunoprecipitation) 32
五、酵素連結免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA) 34
六、生物學路徑分析軟體暨生物資訊資料庫 (Ingenuity Pathway Analysis, IPA; Ingenuity Systems, USA) 35
七、統計分析 35
第四章、結果 37
一、轉譯後修飾 37
二、西方墨點法與免疫沉澱法之驗證 37
三、自體抗體檢測分析 38
四、生物學路徑分析軟體暨生物資訊資料庫分析 38
五、勝算比(Odds Ratio) 39
第五章、討論 40
第六章、結論 45
第七章、文獻資料 47
附圖 49
附表 57
附件 69
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10.Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970. 227(5259): p. 680-5.
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