利用質譜儀與表面電漿共振儀於自體免疫性疾病之生物標誌之探討與研究

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陳昱任(Yu-Ren Chen) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

利用質譜儀與表面電漿共振儀於自體免疫性疾病之生物標誌之探討與研究
(Investigation on the Biomarkers of Autoimmune Disease by Mass Spectrometry and Surface Plasmon Resonance)

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摘要(中)

生物標誌(biomarker)指利用生物分析方式來測量正常人體生理反應、疾病發展過程、藥物生理反應及藥物安全性等，同時可以提供做為臨床上決策依據之生物特徵。生物標記的應用包括了解致病原因、早期發現疾病、追蹤治療的效果及發展新藥。
因此本研究利用了質譜儀與表面電漿共振儀分別對類風濕關節炎及紅斑性狼瘡進行生物標誌相關之研究，主要可分為兩大主題，一為利用質譜儀探尋使用抗腫瘤壞死因子及anti-CD20抗體生物製劑能有效治療類風濕關節炎之生物標誌，主要是想利用質譜儀結合Fe3O4/TiO2磁性奈米粒子，於使用不同生物製劑之病人血清中找尋出新的生物標誌；二為利用表面電漿共振儀量測紅斑性狼瘡病人之人體血清中抗雙股去氧核醣核酸抗體之世代研究，主要於生物晶片改質上雙股去氧核醣核酸，並結合本實驗室發展之表面電漿共振儀，針對已知之生物標誌(抗雙股去氧核醣核酸抗體)去進行檢測，並與現行方式酵素連結免疫分析法(ELISA)去進行比較。
在實驗結果方面也是分兩部分去進行探討，在第一部分方面，成功利用Fe3O4/TiO2磁性奈米粒子提高質譜訊號之表現，獲得了更佳的信號強度，並發現了3.89 kDa與7.77 kDa之標誌，在使用Enbrel此種生物製劑之病人中，3.89 kDa所得之Specificity和Sensitivity可達75%和83%，而7.77 kDa所得之Specificity和Sensitivity可達75%和79%；在使用Humira此種生物製劑之病人中，7.77 kDa所得之Specificity和Sensitivity可達70%和75%。在第二部分方面，本研究計畫為一世代研究計畫，利用自組裝層膜技術及結合EDC/sulfo-NHS改質方式，並將雙股去氧核醣核酸固定化於晶片表面，成功地建立了一個檢測去氧核醣核酸抗體之平台，目前已於壢新醫院收集13名紅斑性狼瘡病患，在不同收集時間內，共得24名病患血清，同時也獲得4名健康人之血清作為對照組，經由ROC曲線圖與p值去進行辨別，顯示出SPR對此抗雙股核醣核酸抗體之辦別能力較ELISA好，也顯示出SPR之診斷價值較ELISA佳。之後也將與壢新醫院持續進行合作，持續地收集病人檢體，進行前瞻性長期觀察之研究，之後會將每個月門診追蹤病情之病人數據整合起來，建立一個以時間軸導向的變化圖，期望最終能建立一具足夠敏感度(sensitivity)及專一性(specificity)之SPR量測血清中抗雙股去氧核醣核酸抗體之檢測平台，及早期檢測與臨床判定之有效方法，此一發展為現今生物醫學工程發展之重點之一，也是轉譯醫學之重要一環。

摘要(英)

Biomarker is a biologic analytical method to measure the normal physiological reaction, to understand the pathology, and drug safety. Besides, biomarker can be a characteristic for decision making in clinical diagnosis. The applications of biomarker include etiopathology understanding and the effect of follow-up care and the drug discovery.
In this study, we investigated the biomarkers to rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) detected by mass spectrometry (MS) and surface plasmon resonance (SPR), respectively. There are two topics involved in this research. First one is to obtain the biomarkers for anti-tumor necrosis factor (anti-TNF) and anti-CD20 which are the possible treatment to RA using mass spectrum. In this topic, we combined MS with Fe3O4/TiO2 nano-particle to search for the new biomarkers applied to different biologics from patient serum. The other topic is a cohort study on detecting anti-double strained deoxyribonucleic acid antibody (anti-ds DNA antibody) from the serum of SLE patient. We used gold chip immobilized with dsDNA to detect anti-dsDNA antibody by our lab-owned SPR, and compared it with enzyme-linked immunosorbent assay (ELISA) on the sensitivity and specificity.
From the results of first topic, we successfully enhanced the detection signal performance of MS by Fe3O4/TiO2 nano-particle and found out two biomarkers with molecular weight of 3.89 kDa and 7.77 kDa. In specific, from the result treated with Enbrel, the specificity using 3.89 kDa or 7.77 kDa biomarker approximates to 75%. And the sensitivity using 3.89 kDa and 7.77 kDa biomarkers approximate to 83% and 79%, respectively. Besides, from the result treated with Humira, the sensitivity and specificity using 7.77 kDa are 70% and 75%, respectively.
The second topic is a cohort study, that is, a detection platform establishment to detect anti-ds DNA antibody with the sensor chip which synthesized by mixed self-assembling membrane (SAM) technique and EDC/sulfo-NHS surface modification. We have collected the information from thirteen SLE patients, obtained the serum samples from 24 patients in different time and the controlling variables from 4 normal people.
From the receiver operating characteristic (ROC) curve and the
p value measurement, we found that the recognition efficiency of dsDNA to anti-dsDNA antibody from SPR detection is better than that of from ELISA, so does the diagnose value. In general, we can successfully establish a detection platform with high sensitivity and specificity to detect the anti-dsDNA from serum and provide the guidance for early diagnosis in the clinical medicine, which is a significant achievement in nowadays translational medicine.

關鍵字(中)

★ 質譜
★ 類風濕性關節炎
★ 表面電漿共振
★ 系統性紅斑性狼瘡

關鍵字(英)

★ Mass Spectrometry
★ Rheumatoid Arthritis
★ Surface Plasmon Resonance
★ Systemic Lupus Erythematosus

論文目次

中文摘要 i
Abstract iii
目錄 v
圖目錄 ix
表目錄 ix
第一章緒論 1
1.1研究背景 1
1.2研究動機 3
1.3研究方法與架構 5
第二章文獻回顧 7
2.1生物標誌 7
2.2自體免疫疾病 9
2.2.1類風濕性關節炎 11
2.2.1.1類風濕性關節炎在臨床上之診斷 12
2.2.1.2類風濕性關節炎之血液檢測 13
2.2.1.3類風濕性關節炎治療方式之發展 15
2.2.1.4生物製劑之發展 16
2.2.2系統性紅斑性狼瘡 19
2.2.2.1系統性紅斑性狼瘡於臨床上之診斷 19
2.2.2.2系統性紅斑性狼瘡之血液檢測 20
2.2.2.3 Anti-dsDNA抗體現行之檢測方式 22
2.3基質輔助雷射脫附離子化質譜儀 25
2.3.1基質的特性及作用 27
2.3.2基質輔助雷射脫附離子化法的離子化機制 29
2.3.3基質輔助雷射脫附離子化法的優缺點 30
2.3.4蛋白質於質譜儀之檢測應用 31
2.4 表面電漿共振儀 34
2.4.1表面電漿現象原理 35
2.4.3表面電漿共振影像儀 44
2.4.4 晶片改質 49
2.4.4.1自組裝單層膜 49
2.4.4.2表面分子之固定化 52
第三章實驗材料與方法 56
3.1實驗藥品 56
3.2實驗設備 59
3.3 實驗方法 60
Part 1探尋使用抗腫瘤壞死因子及anti-CD20 抗體生物製劑能有效治療類風濕關節炎之生物標誌 60
3.3.1 Fe3O4/TiO2奈米粒子之合成 60
3.3.2血清結合Fe3O4/TiO2奈米粒子之處理 61
3.3.3 SDS-PAGE之操作步驟 64
Part 2利用表面電漿共振量測紅斑性狼瘡病人之人體血清中抗雙股去氧核醣核酸抗體之世代研究 65
3.3.4緩衝溶液之配製 65
3.3.5 SPR晶片製備 65
3.3.6 SPR晶片之表面改質 65
3.3.7 ESCA實驗 67
3.3.8血清之前處理 67
3.3.9抗雙股去氧核醣核酸抗體於表面電漿共振儀之檢測 68
第四章結果與討論 68
Part 1探尋使用抗腫瘤壞死因子及anti-CD20抗體生物製劑能有效治療類風濕關節炎之生物標誌 68
4.1病人基本資訊 68
4.2 Fe3O4/TiO2奈米粒子之角色 69
4.3 質譜分析之結果 71
Part 2利用表面電漿共振量測紅斑性狼瘡病人之人體血清中抗雙股去氧核醣核酸抗體之世代研究 79
4.4表面探針固定化鑑定 79
4.5表面不同比例之硫醇分子對吸附量之影響 84
4.6Anti-dsDNA抗體專一性之探討 86
4.7 SPR檢測之結果與比較 87
4.8 ROC曲線及統計分析意義 89
4.9 動力學之分析 94
第五章總結論 98
第六章未來展望 100
參考文獻 101

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2013-8-28

推文