不論良性及惡性的膽道疾病皆可造成膽道阻塞之症狀，但現今對於鑑別良性與惡性膽道疾病仍是一個很大的難題。膽道癌的病人求診時大都已錯過治療的時機，而想利用手術切除已侵犯到血管及膽道的癌組織是不可能的，所以找尋能早期診斷膽道癌的生物標誌是非常重要而迫切的。過去有許多生物標誌被利用來鑑別診斷膽道阻塞之原因，但是這些標誌的靈敏度及特異性均不夠好。為了提供早期正確的診斷及評估病人的預後，也需要新的生物標誌來幫助。蛋白質的硝化，被認為是一種轉譯後修飾作用，通常為發炎時產生的硝化壓力所導致。硝化壓力過去已被報導在許多疾病形成過程中扮演很重要的角色。本篇研究利用蛋白質體學之技術，建立不同膽道疾病的膽汁蛋白質圖譜，並探討可作為鑑別診斷之生物標誌及膽汁內硝化蛋白。藉著混合9位膽結石及膽道癌病人的膽汁，並先行去除膽汁內大量的白蛋白及脂質以得到更好解析度的圖譜。最後，利用二維電泳的技術成功地建立膽汁的蛋白質圖譜。在硝化蛋白質體學方面，我們先利用化學方法合成過氧亞硝基物，使其與蛋白質作用形成硝化蛋白，再注入兔子使其產生硝化酪胺酸抗體並檢測其效價。同時也利用毛細管電泳建立硝化酪胺酸的檢測。膽汁中的硝化蛋白質的偵測方法主要是結合二維電泳分析及硝化酪胺酸抗體西方墨點法。最後，對於在二維電泳分析膠體上有興趣的蛋白質點，利用質譜儀鑑定身分。硝化血紅素結合蛋白的功能分析，主是利用西方墨點法及增強性冷光聚丙烯胺膠體電泳方法進行研究，並進一步分析膽道疾病病人之血紅素結合蛋白型別分佈。本研究結果，分析比較不同族群的膽汁蛋白質圖譜差異性之表現，發現2個在膽道癌中較大量表現之蛋白質點，由質譜儀鑑定結果為elastase 3B。在膽道疾病病人膽汁內之硝化蛋白，發現有4個硝化酪胺酸陽性的蛋白質點在膽結石及膽道癌中皆有表現，但膽道癌量較少，從中選取3個蛋白質點由質譜儀鑑定，結果為人類血紅素結合蛋白。具有硝化酪胺酸之血紅素結合蛋白的功能分析中，發現硝化酪胺酸主要出現於β以及α2鏈；而具有硝化酪胺酸之血紅素結合蛋白，其與血紅素結合之能力也隨之下降。另一方面，分析血紅素結合蛋白型別在不同疾病分佈，膽結石族群與膽道癌相比較，其血紅素結合蛋白為2-1型及2-2型比例較高，且具有統計上意義(96% versus 86%, p<0.05)。總結來說，本研究建立了分析人類膽汁之二維電泳蛋白質圖譜，在膽道癌的膽汁中找到兩個具有潛力之生物標誌 - elastase 3B及硝化血紅素結合蛋白。Elastase 3B在膽道癌所扮演的角色需要進一步的確認及探討其功能。膽汁內血紅素結合蛋白在膽結石扮演重要之角色，可能藉由與血紅素結合能力改變所導致，而其所產生硝化酪胺酸之現象，顯示一氧化氮所衍生出的活性氮物質可能在膽道疾病扮演角色，而這樣的結果也將硝化壓力、血紅素代謝、膽道疾病之間的關係相連接。

　Both benign and malignant biliary tract disease can cause biliary obstruction. The differential diagnosis between them is difficult as the neoplastic foci may be trivial and combined with other benign diseases. Early detection of biliary cancers is important for the survival of patients as the advanced biliary cancers usually involve the bile ducts and vessels, which render the surgical resection impossible. Various markers have been used for differential diagnosis of biliary obstruction. However, they are neither sensitive nor specific. A novel marker with a better diagnostic efficacy is therefore needed. Nitration of proteins, a post-translational modification of proteins resulting from nitrosative stress, may play a role in the pathogenesis of various diseases. The aim of this study is to construct the maps of biliary proteome in order to search new biomarkers for differential diagnosis and studying pathogenesis of various biliary tract diseases by differential proteomic analysis and nitrotyrosine immunoblotting. Biliary proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Composite samples were prepared by pooling biles from nine gallstone and nine biliay tract cancer patients. The albumin and lipid were removed before analysis to reduce the interference and achieve a better resolution. For the nitroproteome study, peroxynitrite, prepared by chemical reaction, was mixed with KLH to form nitrated proteins to immunize rabbits. Nitrotyrosine antibodies were expressed by rabbit immunization. A capillary electrophoresis method was developed for validating the presence of 3-nitrotyrosine in nitrated proteins. The nitrated proteins in bile were visualized by 2D-PAGE-based western blotting. The interesting protein spots were identified by mass spectrometry. The functional analysis of nitrated haptoglobin was done by western blot and enhanced CL PAGE analysis. The haptoglobin phenotype of patients was also determined by enhanced CL PAGE analysis. By comparing the proteomic maps of different groups, two protein spots of elastase 3B were found highly expressed in the biliary cancer group. Three nitrated proteins were found less expressed in the biliary cancer group, and were matched to haptoglobin. The sites of nitrotyrosine in haptoglobin were located in haptoglobin β and α2 chains. The binding activity of haptoglobin-hemoglobin was decreased in nitrated haptoglobin. In analysis of haptoglobin phenotype distribution, the gallstone group had significantly higher Hp2 carrier than cholangiocarcinoma group (96% versus 86%, p<0.05). In conclusion, the map of bile proteins has been successfully established. Two candidates - elastase 3B and nitrated haptoglobin were found by proteomic analysis. Our observations suggest that elastase 3B may be a biomarker for biliary cancers and further study is needed to validate its clinical use. The detection of nitrate haptoglobin in bile suggest that reactive nitrogen species derived from nitric oxide might involve in the pathogenesis of biliary tract diseases. The studies of nitrated haptoglobin might be able to link nitrosative stress, hemoglobin metabolism, and biliary tract disease together.

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