現今對於鑑別良性與惡性膽道疾病仍是一大難題。過去的研究顯示，許多膽汁中的腫瘤標誌靈敏度及特異性均不夠好。在我們實驗室，曾利用蛋白質體學的技術，發現膽道癌病人膽汁中胰臟彈性蛋白酶3B有大量表現。胰臟彈性蛋白酶(PE)是一種內胜肽酶分子量為29 KDa，是屬於絲胺酸蛋白酶的家族成員之一，可水解許多細胞外的基質，例如不可溶彈性蛋白、纖維蛋白、醣蛋白以及血色素。然而近年來有研究指出，PE可誘發黏液大量分泌及在發炎所造成的組織傷害中扮演重要的角色。但目前對於PE大部份的生物特性並不十分清楚。因此本研究將探討PE在正常和癌化的人組織、癌化細胞株及膽道疾病病人膽汁的分佈，發展測定膽汁PE活性的方法，探討其臨床應用性；並進一步確效PE在惡性膽道疾病所扮演的角色。組織萃取液分別利用定量聚合酶儀及西方墨點法測定mRNA及蛋白質的表現量。膽道阻塞的病人利用膽道引流術抽出膽汁。這些病人的診斷包括膽結石(n=29)、膽道癌(n=22)及胰臟癌(n=23)。利用分子模版轉印聚合物去除膽汁干擾物，再利用合成受質測定PE的活性。在TFK-1細胞株加入PE，並利用ELISA測定TNF-α的表現。結果顯示在人類肝癌細胞株Huh7、Hep3B、HepG2和人類膽道癌細胞株TFK-1及膽道癌病人有癌化的肝臟中皆有ELA3B mRNA和蛋白質的表現。膽汁中PE蛋白質表現的陽性率，在惡性膽道疾病族群比膽結石族群較高（46% vs 11%；chi-square,p<0.005)。若分析膽道癌族群膽汁中PE蛋白質表現陽性率則更加明顯（63%）與膽結石族群相比較(p<0.0005)。利用膽紅素特異的分子模版轉印聚合物前處理膽汁後，再進行PE活性的測定，此分析方法之再現性(CV＜7.3%),平均回收率92% (範圍, 86%~103%)。惡性膽道疾病病人膽汁之PE活性較良性膽道疾病者高(16.2 U/L vs. 6.4 U/L, p<0.01)。為了排除胰液逆流至膽汁造成PE活性的上升，利用膽汁澱粉酶的測定。結果顯示，膽汁內PE/amylase比值在惡性膽道疾病族群 (0.228 ± 0.043)較膽結石族群(0.027 ± 0.006)更為顯著高(p<0.0001)。在ROC曲線圖下的面積，PE/amylase比值(決定值0.065)在診斷惡性膽道疾病正確性為0.874,靈敏度78.6%和特異性86.2%。添加PE於TFK-1細胞株3小時即可顯著誘發TNF-α表現。總結來說，本研究指出PE在惡性疾病有較高的表現及在膽道疾病病程中扮演重要的角色。測定膽汁PE/amylase比值可做為鑑別診斷膽道阻塞的生物標誌。

It is very difficult to differentiate benign from malignant cause of biliary obstruction. Many tumor markers in bile have been reported attemping to help diagnose malignancy biliary tract diseases. However, none of these markers exert satisfactory sensitivity and specificity. Previous study in our laboratory, using proteomics approach for a global analysis of human bile, found that pancreatic elastase 3B was highly expressed in patients with malignancy. Pancreatic elastase (PE), a 29 KDa endopeptidase, is a family of serine protease, which degrades extracellular matrix components, such as insoluble elastin, fibronectin, proteoglycan, and hemoglobin. Recent studies indicate that PE induces hypersecretion of mucin and plays an important role in inflammatory-mediated tissue injury. Nevertheless, most of biological properties of PE remain uncleared. The aim of this study is to determine the distribution of PE in normal and malignant tissues, cell lines, and bile from patients with biliary obstruction, to establish and validate an assay for the measurement of PE activity in bile and its clinical application, and to characterize the role of PE in the biliary malignancy. Levels of mRNA and protein expression were determined by real-time PCR and western blotting, respectively. Bile samples were collected from patients with bile duct obstruction on the day of biliary drainage. The etiology of biliary obstruction includes gallstone (n=29), cholangiocarcinoma (n=22), and pancreatic cancer (n=23). A bilirubin-specific molecularly imprinted polymer (MIP) was used as a bioseparator to extract bile proteins for the determination of PE activity. PE was added in TFK-1 cell cultures and evaluated its TNF-α expression by ELISA. The results showed that the expression of ELA3B mRNA is up-regulated in Huh7, Hep3B, HepG2, and TFK-1 cell lines, and in the tumor part of liver from patients with cholangiocarcinoma. PE protein expression was also observed in Huh7, Hep3B, HepG2, and TFK-1 cell lines. The positive rate for PE protein detected by western blotting was increased in bile samples from patients with biliary malignancy, compared to those with gallstone（46% vs 11%, chi-square, p<0.005). The positive rate for PE protein expression in the bile was much higher in patients with cholangiocarcinoma, than those patients with gallstone（63% vs 11%，p<0.0005). After pretreatment with MIP, PE assay of bile has an average recovery of 92% ( range, 86%103%) and the CV＜7.3%. The activities of PE in bile were significantly higher in patients with biliary malignancy (16.2 U/L vs. 6.4 U/L, p<0.01). Using biliary amylase to exclude the existence of pancreaticobiliary reflux, the ratio of biliary PE to amylase was markedly elevated in patients with malignancies (0.228 ± 0.043), compared to those with gallstone (0.027 ± 0.006, p<0.0001). The area under the ROC curve of biliary PE/amylase for the diagnosis of malignant obstruction was 0.874, using a cutoff value of 0.065. The sensitivity and the specificity for biliary PE/amylase ratio were 78.6% and 86.2%, respectively. Treatment of TFK-1 cells with PE resulted in an increased level of TNF-α. PE-induced TNF-α secretion in TFK-1 cell line reached its peak at 3hrs (p<0.0001). In conclusion, our data indicate that the expression of PE was higher in malignancy and may play a role in the pathogenesis of biliary malignancy. Biliary PE/amylase ratio could be a useful marker in the differential diagnosis of biliary obstruction.

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