以蛋白質體學方法鑑定人類肺腺癌之血清生物標記

Abstract

肺癌為近年來台灣地區癌症造成死亡原因的第一名，且死亡率逐年攀升，據衛生署的統計，民國94年台灣地區有7302人死於肺癌，超過所有因癌症死亡人數的五分之一。肺癌可分成兩大類型：小細胞肺癌與非小細胞肺癌，非小細胞癌可再分成鱗狀細胞癌，腺癌與大細胞癌等。由於肺癌具有不易早期發現，惡性程度高，易轉移，死亡率高等特性，因此盡早檢測肺癌的發生，對於肺癌病人的治療，有非常重要的幫助。 本論文的主要研究目標為利用蛋白質體學的技術，尋找肺腺癌病人血清中具潛力的生物標記 (biomarker)。利用二維電泳比較肺腺癌病人與正常人之血清蛋白質，我們發現fibrinogen α、fibrinogen β、fibrinogen γ、alpha-1 antitrypsin、serum amyloid A (SAA) 等五個蛋白質，在肺腺癌病人的血清中，含量明顯高於正常人。透過西方墨漬法(Western blotting)，也證實隨著期別的增加，血清中含serum amyloid A (SAA)蛋白的肺腺癌病人比例有上升的趨勢。同樣利用二維電泳比較肺腺癌病人肺部癌化組織與正常組織蛋白質的差異，找出在癌化組織表現量較高的蛋白質群，並利用分析軟體與文獻的搜尋，評估其中可能為分泌性蛋白 (secreted protein) 的蛋白質，我們發現了galectin-3、galectin-1、peroxiredoxin、triosephosphate isomerase (TIM)、glyceraldehyde phosphate dehydrogenase (GAPDH)、pyruvate kinase (PK)、phosphateglycerate kinase 1等七個蛋白質，在癌化組織中的量高於正常組織，並可能由組織分泌或流失至血中。透過ELISA的實驗也驗證了galectin-3蛋白在肺腺癌病人的血清中，含量的確較正常人高。最後，我們在肺腺癌病人的肺部癌化組織中，發現了E-cadherin被蛋白酶切割的現象，並偵測到在肺腺癌病人的血清中，E-cadherin蛋白片段(fragment)的量高於正常人。 本實驗室未來的研究，將繼續找尋肺腺癌病人血清中其它蛋白質生物標誌，最後希望能夠利用這些具有潛力的生物標誌，建立複合型生物標誌(multiple biomarkers)，作為肺腺癌早期檢測或預後評估的指標。

Lung cancer is the leading cause of cancer death in Taiwan for recent years, and the mortality rate is increasing annually. According to the statistic report of Department of Health, 7302 people was died from lung cancer in Taiwan in 2005, which over one-fifth of all cancer deaths number. Lung cancer is generally divided into 2 types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Non-small cell lung cancer can divided into three subtype including squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Due to the symptoms of delayed-diagnosis, malignancy, metastatic capacity and highly-mortality rate, early detection of lung cancer could be great help in patient treatment . The major interest of this study is to find out the potential biomarkers existing in the sera of lung adenocarcinoma patients through proteomics approaches. Compare the protein profile between the sera of lung adenocarcinoma patients and normal volunteers by 2D-electrophoresis, expression of fibrinogen α, fibrinogen β,fibrinogen γ, alpha-1 antitrypsin and serum amyloid A (SAA) are elevated in sera of lung adenocarcinoma patients. Increasing SAA concentration was also observed in the blood of late-staged lung adenocarcinoma patient through the confirmation of western blotting. Meanwhile, by comparison of the 2DE protein profile between lung cancerous tissue and non-cancerous tissue of lung adenocarcinoma patient , seven potential secreted proteins including galectin-3, galectin-1,peroxiredoxin 1,triosephosphate isomerase (TIM), glyceraldehyde phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), and phosphateglycerate kinase were discovered based on software analysis and literature research. ELISA result showed that there was increasing galectin-3 in the serum of lung cancer patient compared with the normal volunteer. Finally, cleavage of E-cadherin in cancerous tissue and the elevated expression of cleaved E-cadherin fragment in the serum of lung adenocarcinoma patients was observed. Further confirmation of the finding in this research and discovery of other potential secreted proteins expressed specifically in lung cancerous tissues is needed. Hopefully, we can construct these potential biomarkers into a multiple biomarkers panel , and look forward to using as diagnostic and prognostic tool for lung cancers in the future.