- 學位論文
以蛋白質體學方法鑑定與人類肝細胞癌相關之腫瘤標記
Identification of human hepatocellular carcinoma-related biomarkers using proteomic approaches
摘要
肝細胞癌（簡稱肝癌; HCC）是全世界常見的惡性腫瘤之一，其發生的主要原因與B型肝炎病毒、C型肝炎病毒的慢性感染有關，其他可能的因素包括酒精和食物遭黃麴毒素的污染。自從西元1984年至今，肝癌就高居台灣十大癌症死因的首位，每年大約有六千至八千個國人死於肝癌，雖然目前以超音波及甲型胎兒蛋白定期追蹤檢查，可以早期篩檢出肝癌，然而肝癌的治療仍不理想。預後不良的原因，除了發現太晚之外，肝癌復發也是一個很重要的原因。因此如果想要提升肝癌的治療效果，進一步研究肝癌的致癌機轉及找尋更好的早期診斷標記仍是必須的。在本論文第一個部分中，我們利用螢光二次元差異性電泳 (2D-DIGE) 合併液相層析偶合串聯式質譜儀 (nanoLC-MS/MS) 來分析肝癌組織與非肝癌組織蛋白質表現量的差異。我們希望藉由此技術，可以找到跟肝癌發生相關的蛋白質生物標記，這些與肝癌相關的蛋白質可能具有潛力可作為日後早期診斷肝癌或肝癌治療的標記。在八個肝癌病人手術檢體中，分別對肝癌組織與非癌肝組織所萃取出的蛋白質標示Cy3-和Cy5-螢光染劑並同時對一個等量的內部控制樣品 (八個肝癌組織與八個非肝癌組織所萃取出的蛋白質混和樣品) 標示Cy-2螢光染劑，將這三個螢光標示樣品混和後進行第一維等電點聚焦電泳 (IEF) 和第二維SDS 聚丙烯醯胺膠體電泳 (SDS-PAGE)分析。結果顯示三十四個蛋白特徵點有表現量顯著差異(配對t檢定，p< 0.05)並鑑定分屬三十種不同的蛋白。其中十六個在肝癌組織中表現量上升而另十四個在肝癌組織中表現量下降，這些蛋白似乎在許多細胞分子調控路徑上扮演重要角色，包括醣解、脂肪酸運輸及交流、胺基酸代謝、鐵及外來物代謝、酒精代謝、細胞週期調控、細胞骨架合成和壓力。我們發現14-3-3γ這個蛋白在肝癌組織中表現量顯著上升，而14-3-3異構物 (isoform) 已被証實和癌化有關，因為他們參與調控細胞增生、自殺及分化等路徑。綜合本實驗之結果，証明螢光二次元差異性電泳為一有效的技術使我們能鑑定出在肝癌細胞中表現具差異性的蛋白，鑑定這類有潛力的生物標記將有助於對肝癌化機制進ㄧ歩的了解。 第二部份則是利用蛋白質晶片及表面增強雷射去脫附/游離飛行質譜儀(surface-enhanced laser desorption/ionization time-of-flight mass spectrometry)來尋找肝癌病患血清中跟癌化有關的生物標記。我們總共收集55個肝癌血清檢體、48個慢性肝炎血清和9個健康對照組血清，並利用具弱陽離子交換2型蛋白晶片(Weak Cation Exchange 2)及SELDI-TOF MS來研究肝癌血清中低於20 kDa分子量蛋白差異表現。我們發現一個位於8.9 kDa附近的蛋白在慢性C型肝炎及C型肝炎病毒相關肝癌病患中有明顯上升的表現。進ㄧ步利用二次元膠體電泳及液相層析偶合串聯式質譜儀鑑定此蛋白為補體C3a (complement C3a)。補體C3a在肝癌血清中表現也被我們用蛋白晶片免疫分析法及西方墨點法證實。本篇研究的主要貢獻除了發現補體C3a與C型肝炎病毒所引起的肝炎和肝癌相關外，合併蛋白質晶片技術和二次元膠體電泳及串聯式質譜將提供找尋疾病相關生物標記時一個解決的辦法。 綜合本研究之實驗結果，證明以蛋白體方法學尋找肝癌的生物標記是一個有效的策略。未來，我們將繼續從肝癌的組織及血清中研究疾病相關分子在轉譯後修飾層級的變化。期望從這樣的研究中，找到多個癌症相關的標記，進而建立肝癌多標記診斷蛋白晶片系統，這將有助於了解肝癌的致病機轉及幫助臨床早期診斷。
並列摘要
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death throughout the world. Although hepatitis B or C viral infections are main risk factors for HCC, the molecular mechanisms leading to HCC formation have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. In this study, we employed two-dimensional difference gel electrophoresis (2D-DIGE) combined with nano flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) to investigate differentially expressed proteins in HCC. For each of eight HCC patients, Cy3-labeled proteins isolated from tumor tissue were combined with Cy5-labeled proteins isolated from the surrounding nontumor tissue and separated by 2D gel electrophoresis along with a Cy2-labeled mixture of all tumor and nontumor samples as an internal standard. Thirty-four protein spots corresponding to 30 different proteins were identified by nanoLC-MS/MS as showing significant change (paired t-test, p < 0.05) in the level of expression between tumor and nontumor tissues. Sixteen proteins were up-regulated and 14 were down-regulated in HCC; they seem to play important roles in a variety of pathways including glycolysis, fatty acid transport and trafficking, amino acid metabolism, iron and xenobiotic metabolism, ethanol metabolism, cell cycle regulation, cytoskeleton, and stress. A remarkable finding is the up-regulation of 14-3-3γ protein in HCC. 14-3-3 isoforms had been linked to carcinogenesis because they are involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation, and differentiation. In conclusion, 2D-DIGE is an efficient strategy that enables us to identify differentially expressed proteins in HCC. Identification of potential biomarkers, such as the pinpointing of 14-3-3γ in our findings, may provide further useful insights into the pathogenesis of HCC. The second part of our work is using the ProteinChip technology to search HCC-related serum biomarkers. We performed surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify differentially expressed proteins in HCC serum using weak cation exchange (WCX2) protein chips. Protein characterization was performed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation and nano flow liquid chromatography tandem mass spectrometry (nano LC MS/MS). A total of fifty-five sera were collected from HCC patients and compared with forty-eight patients with chronic hepatitis and nine healthy individuals. A candidate marker with molecular weight about 8900 Da was detected as differentially expressed in patients with chronic hepatitis C and hepatitis C virus-related HCC. We identified this differentially expressed protein as complement C3a. The expression of C3a in HCC sera was further validated by PS20 chip immunoassay and Western blotting. Complement C3a was found to be elevated in patients with chronic hepatitis C and HCV-related HCC. Combination of SELDI-TOF MS and 2D-PAGE provide a solution to identify disease-associated serum biomarkers. Taken together, our results show using proteomic approaches to identify HCC biomarkers is an efficient strategy. In the future, we will investigate the posttranslational modification changes of HCC-associated biomarker in the tumor tissues and sera, and set up the multimarker diagnostic protein chip. This will improve our understanding of hepatocarcinogenesis and early detection of HCC.
參考文獻
延伸閱讀
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