大腸直腸癌早期檢測之新穎性血漿刀豆素A吸附蛋白質生物標記的開發計畫

Taipei Medical University Institutional Repository > 醫學科技學院 > 醫學檢驗暨生物技術學系所 > 博碩士論文 > Item 987654321/54092

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題名:	大腸直腸癌早期檢測之新穎性血漿刀豆素A吸附蛋白質生物標記的開發計畫 The development plan of plasma novel Con A-bound protein biomarkers for early detection of colorectal cancer
作者:	黃秀雯 Huang, Hsiu-Wen
關鍵詞:	大腸直腸癌;生物標記;colorectal cancer;biomarker
日期:	2013-07-19
上傳時間:	2018-11-16 12:21:12 (UTC+8)
摘要:	大腸直腸癌為全世界常見的癌症之一，在台灣大腸直腸癌的發生率高居第二位，死亡率排名第三位。目前大腸直腸癌篩檢的方法有大腸直腸內視鏡、糞便潛血篩檢及生物標記篩檢。大腸直腸內視鏡為侵入式的檢查，病人容易排斥此類型的檢查；糞便潛血篩檢雖然方便但其偽陽性的機率較高；臨床上最常使用的生物標記為腫瘤胚胎抗原（CEA）以及癌抗原 19-9 (CA 19-9)，雖然為非侵入性的檢查方法，但準確率及靈敏度不高是其缺點。而蛋白質轉譯後修飾在生物體內扮演重要的角色，目前已知癌症及自體免疫性疾病的病人，其體內蛋白質的轉譯後修飾會產生變化，因此本研究致力於開發血漿蛋白質轉譯後修飾相關的生物標記，我們以蛋白質體分析策略，透過比較健康人及大腸直腸癌患者血漿中，其蛋白質量（表現量）及質（轉譯後修飾）的變化，找出具有大腸直腸癌早期篩檢潛力的生物標記。在血漿樣本中，我們利用醣類凝集素瓊脂純化醣蛋白，並以奈米級液相層析串聯質譜（Nano-LC-MS/MS）進行分析，鑑定血漿中微量蛋白質的變化；以及分析軟體：PTM-Miner 及 PTM-Q，進行蛋白質轉譯後修飾的鑑定及定量分析。我們共鑑定出 161 個蛋白質，及 2741 個轉譯後修飾位點；其中有 15 個蛋白質含量在統計上有顯著差異 (p < 0.05)。以西方墨點法進一步驗證其中四個蛋白質：載脂蛋白 L1 (apolipoprotein L1, Apo L1)、載脂蛋白 E (apolipoprotein E, Apo E)、甲一型抗胰蛋白酶 (alpha-1-antitrypsin, AAT) 以及血小板反應蛋白 (thrombospondin-1, TSP-1)，經過接收操作特徵曲線 (ROC curve) 分析後，全血漿及 Con A 吸附 Apo L1、Con A 吸附 Apo E、全血漿 AAT，這四類蛋白檢測之敏感度為 95.8 %、100.0 %、87.5 %、91.7 %；特異性分別為 100.0 %、95.8 %、 87.5 %、66.7 %，顯示這些蛋白質都具有大腸直腸癌篩檢之潛力。而其中我們鑑定出載脂蛋白 E 以及甲一型抗胰蛋白酶具有新穎性的轉譯後修飾，在將來也可以發展為大腸直腸癌診斷之生物標記。 Colorectal cancer is a common type of cancer all over the world. It is the second prevalent cancer in Taiwan and the third cause of cancer motility. Current colorectal cancer screening is based on colonoscopy, fecal occult blood test, and biomarker analysis. Among these methods, colonoscopy usually requires invasion of tissues and therefore is easily rejected by patients. Fecal occult blood test appears to be the most convenient but frequently exhibits false-positive results. As for the most common biomarkers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9), the analysis is not invasive but has very limited accuracy and sensitivity. An important mechanism of protein post-trasnslational modifications (PTMs) is considered pivotal in biological functions. Studies have shown that patients who carry cancers or autoimmune diseases may generate altered PTM patterns. Therefore, we devote to develop a novel PTM biomarker analysis for colorectal cancer early diagnosis. Our study will apply strategies of proteomic analysis to analyze the plasma samples from normal subjects and colorectal cancer patients. The analysis will distinguish the differences of plasma protein composition between the normal and the patients. We analyze the differences among samples, focused on the level of protein expression (quantitatively) and the type of PTMs (qualitatively). In this study of plasma samples, lectin agarose was integrated to purify glycoproteins. The collected samples were used Nano-LC-MS/MS to identify the lower abundance proteins and then used data analysis softwares (PTM-Miner and PTM-Q) to collect PTM information. We identified 161 protein and 2741 modification target sites. Quantitative statistic results indicated that 15 proteins were analyzed significantly ( p < 0.05). Then, western blot was used to confirm four of these proteins, including apolipoprotein L1、apolipoprotein E、alpha-1-antitrypsin and thrombospondin-1. A subsequent analysis by ROC curve indicated that sensitivity of total plasma Apo L1、Con A bound Apo L1、Con A bound Apo E and total plasma AAT were 95.8 %、100.0 %、87.5 %、91.7 %. The specificity were 100.0 %、95.8 %、 87.5 %、66.7 % separately. Results suggested that these proteins may be potential plasma biomarkers for the detection of colorectal cancer. We also identified that there were novel PTMs on peptide sequences of apolipoprotein E and alpha-1-antitrypsin. Our study showed that in the future we can use such PTM-related peptides as potential biomarkers for clinical diagnosis of colorectal cancers.
描述:	碩士指導教授-林景堉共同指導教授-張世慶委員-陳威戎委員-蘇家玉委員-林榮俊
資料類型:	thesis
顯示於類別:	[醫學檢驗暨生物技術學系所] 博碩士論文

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