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研究生:蔡忠憲
研究生(外文):Chung Hsien Tsai
論文名稱:慢性阻塞性肺病患者自體抗體的篩檢
論文名稱(外文):Autoantibodies screening in patients with chronic obstructive pulmonary disease
指導教授:詹爾昌
指導教授(外文):E. C. Chan
學位類別:碩士
校院名稱:長庚大學
系所名稱:生化與生醫工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:63
中文關鍵詞:慢性阻塞性肺病細胞角質蛋白 18自體抗體
外文關鍵詞:chronic obstructive pulmonary diseasecytokeratin 18autoantibody
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慢性阻塞性肺病係ㄧ種因慢性支氣管炎或肺氣腫所引發,而導致呼吸道氣流阻塞為主要特徵的肺部疾病。其發病率與死亡率很高,預計到2020年時,其死因排名將會升至第五位。雖然已有許多COPD的致病因子被鑑定出來,目前讓肺部及支氣管持續發炎的機制仍不清楚。現今自體免疫反應已被證實參與於COPD致病機轉之中,但是尚未發現和COPD有關的自體抗原和自體抗體。本文中,我們利用西方墨點法分析COPD患者的血清並證實其血清內存在著對抗肺泡上皮細胞抗原的自體抗體。我們發現確實有對抗肺泡上皮細胞抗原的自體抗體存在於患者血清中,其最主要相對應的抗原分子量為98, 86, 75, 66, 51, 45, 和31(kD)。利用免疫沉澱法和質譜儀分析45-kD位置的抗原為人類細胞角質蛋白質18(human cytokeratin 18)。我們發現患者和控制組血清中抗細胞角質蛋白質18自體抗體的盛行率分別為74%和32%。使用酵素免疫分析法來偵測抗細胞角質蛋白質18自體抗體的量發現在患者血清中也遠高於控制組血清,而主要病灶為肺氣腫亦高於病灶為慢性支氣管炎的族群。雖然抗細胞角質蛋白質18自體抗體的量與FEV1 (L)和FEV1 (% 預測值)有相關性,卻和疾病分期並無關聯。這是第一次證實與COPD相關的人類細胞角質蛋白質18自體抗原的存在。
Chronic obstructive pulmonary disease ( COPD ) is characterized by airflow obstruction due to chronic bronchitis or pulmonary emphysema。The morbidity and mortality of COPD is related high. Moreover it is expected to rise from its current position as the fifth most common cause of death,worldwide, by the year 2020。Although some risk factors for COPD have been identified,the mechanism of persistent chronic airway inflammation remains unclear。The involvement of autoimmune responses in the pathogenesis of COPD has been proposed; however,no autoantigens or corresponding autoantibodies associated with COPD have been identified. Here,we analyzed the sera of COPD patients to investigate the possible existence of circulating autoantibodies against the A549 cellular proteins。We showed the existence of autoantibodies against the pulmonary cellular antigens in the sera of COPD patients,and the molecular weights (kD) of the predominant immunoreactive autoantigens were 98,86,75,66,51,45 and 31, respectively。Immunoprecipitation and MALDI-TOF mass spectrometry revealed that the 45-kD target autoantigen was human cytokeratin 18 protein。We showed that the prevalence of anti-cytokeratin 18 autoantibodies in the sera of COPD patients and controls was 74% and 32%, respectively。We used ELISA to detect anti-cytokeratin 18 autoantibody level and also found that the anti-cytokeratin 18 autoantibody level was significantly higher in the COPD group than in the control group and in the predominantly emphysema group than in the predominantly chronic bronchitis group。Although we showed that the anti-cytokeratin 18 autoantibody level was significantly associated with FEV1 (L) and FEV1 (% pred),the presence of the anti-cytokeratin 18 autoantibody was not associated with the GOLD stage of the disease.。This is the first report on the identification of the pulmonary epithelial cytokeratin 18 protein as an autoantigen associated with COPD and the demonstration of the prevalence of anti-cytokeratin 18 autoantibodies in COPD patients。
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長庚大學博碩士紙本論文著作授權書
國家圖書館書授權書.……………...…………………………………...iii
長庚大學授書…...….…………………………………………………...iv
誌謝….……………………..…………………………………………….v
中文摘要……………………..........…………………………………….vi
英文摘要…………………....……….........…………………………….vii
目錄………………..…………………………………………………….iv
圖表目錄……...……………………………………………...…………xii
第一章 簡介………………………………………………………...….1
1.1 慢性阻塞性肺病定義……………………………………………...1
1.2 慢性阻塞性肺病診斷及分期…………………………………..….3
1.3 慢性阻塞性肺病的致病機制……………………………..……….4
1.4 自體免疫假說………………………………………………...........7
第二章 研究目的…………………………………................................12
第三章 材料…………………………………………………………....13
3.1 細胞株及其培養基……………………...………………………....13
3.2 蛋白質定量………………………………….………………...…...13
3.3 蛋白質電泳……………………………………….………………..13
3.4 西方墨點法…………………………………………….…………..14
3.5 銀染…………………………………………….......……………....15
3.6 免疫沉澱法…………………….………………………………......15
第四章 方法………………………………………………….………...16
4.1 細胞培養………………………….……………………………......16
4.2 細胞蛋白質萃取..…………………………...……………...……...16
4.4 蛋白質濃度定量..……………………………………...…...……...16
4.5 蛋白質電泳………………………………………………......….....17
4.6 Coomassie Blue stain …………………...…………………......…...17
4.7 西方點墨法………………………………………………………...17
4.8 免疫沉澱法…………………………………………………....…...18
4.9 銀染……..……………………………………………......………...18
4.10 酵素免疫分析法……………..…………………...………..……..19
4.11 組織檢體與血清…………………..…………………........……...19
4.12 統計分析……………………………………………………..…...19
第五章 結果……………………………………………………………21
5.1篩檢COPD患者血清中是否存在有抗肺臟細胞之自體抗體…......21
5.2分析自體抗體效價與COPD臨床病程的關係………………….....21
第六章 討論…………………………………………………….....…...26
參考文獻…………………………………………………...........……...30































圖表目錄
圖一 western-blot assay偵測研究樣本血清中的抗肺臟細胞蛋白的自體抗體……………………………………………………..…...……….35
圖二 免疫沉澱後免疫複體以12%SDS-PAGE膠分離後之銀染結果.36
圖三 西方墨點法鑑定與篩檢研究樣本血清中之Anti-recombinant human cytokeratin 18 protein自體抗體………………………..………37
圖四 確認尋找ELISA條件之檢體………………………………...….38
圖五 抗原吸附濃度篩選………………………………………...…….39
圖六 血清及二級抗體稀釋倍數篩選……………………………..…..40
圖七 呈色時間篩選………………………………………………..…..41
圖八 西方墨點法比較COPD患者與控制組間自體抗體量的相關性.42
圖九 利用酵素免疫分析法比較COPD患者與控制組間自體抗體量的相關性……………………………………………………..……………43
圖十 利用西方墨點法比較肺氣腫與慢性支氣管炎之COPD患者間Anti-cytokeratin 18 autoantibody 量的相關性………………………...44
圖十一 利用酵素免疫分析法比較肺氣腫與慢性支氣管炎之COPD患者間Anti-cytokeratin 18 autoantibody 量的相關性……………..……45
圖十二 Receiver Operating Characteristics Curve ( ROC curve)分析比較西方墨點法及酵素免疫分析法.……………………………………..46
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