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研究生:陳佳華
研究生(外文):Chen chia hua
論文名稱:利用組織間液模式尋找人類肝癌生物標記候選蛋白質
論文名稱(外文):AN INTERSTITIAL FLUID APPROACH TO DISCOVER BIOMARKER CANDIDATES FOR HUMAN HEPATOCELLULAR CARCINOMA
指導教授:謝森永
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:97
中文關鍵詞:肝癌生物標記組織間液蛋白質體學
外文關鍵詞:HCC biomarkerinterstitial fluidproteomics
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癌症生物標記(cancer biomarker)是臨床上作為疾病早期治療、新藥開法以及治療策略…等目的之醫療利器。蛋白質體學技術是現今臨床醫學上找尋癌症生物標記的重要應用之一。在臨床診療上若能有效利用肝癌生物標記以助肝癌早期發現,則可期待肝癌病患存活率的增加。目前臨床上最常被用來做為診斷與偵測的肝癌標α-fetoprotein (AFP)雖具有高度肝癌專一性,但其早期偵測效果卻十分有限,所以尋找兼備高專一性與高靈敏度的新肝癌標記便成為臨床診斷上的需求。本實驗收集九位病患的肝癌及其周邊非腫瘤臨床檢體組織(tissue lysate)以及組織間液(interstitial fluid),利用二維電泳(two dimensional gel electrophoresis,2-DE)以及基質輔助雷射脫附游離(MALDI-TOF)的實驗方法分析蛋白質表現概況,並尋找在肝癌與非肝癌中表現量相異的蛋白質。實驗結果發現PRDX1、CyPA、ERp57、RBP4以及14-3-3 sigma這五個蛋白質在腫瘤周邊體液(TIF)有較非腫瘤周邊體液(NIF)上升的現象,其中CyPA可有效區分肝硬化與正常臨床病患血清,因此有潛力作為肝硬化生物標記。其他四個蛋白質則視為肝癌生物標記候選蛋白質。
One of the main missions of clinical onco-proteomics is not only to discover more biomarkers for early stage diagnosis of disease but also to provide potential targets for drug discovery and therapeutic intervention. According to epidemiology studies, HCC has been the major leading cause of cancer death in the world. It has been widely known that HCC is strongly correlated to HBV/HCV infection and other causalities, however, the molecular mechanisms of hepatocarcinogenesis are still unclear. High sensitivity and specificity HCC biomakers are useful in HCC early detection and hopefully can increase patients’ survival rate. The markers, such as a-fetoprotein, used so far have limited utility when detecting early stage HCC. Therefore, finding novel markers with higher sensitivity and specificity is urgently needed. We collected the tumor (TL) and nontumor (NL) tissue lysate and also the interstitial fluid perfusing the HCC microenvironment from the surgical resects of the tumor (TIF) and para-tumor liver tissues (NIF) in nine cases of hepatoma, and analyzed them using 2-DE and MALDI- TOF MS. The amount of five proteins- PRDX1, CyPA, ERp57, RBP4 and 14-3-3 sigma were frequently found to be higher level in TIF as compared to those in the NIF. The results were further verified via immunoblotting analysis. HCC and cirrhosis patients’ serum have higher cyclophilin A level than normal ones. Our findings suggest that these five proteins, particularly cyclophiln A, could be potential markers for diagnosis of HCC.
第一章 前言
1. 1 肝癌簡介……………………………………………. 1
1. 2 肝癌致病因子………………………………………. 1
1. 2. 1 病毒性致病因子……………………………………. 2
1. 2. 2 酒精性與黃麴毒素-B1致病因素………………….. 3
1. 3 肝癌臨床檢測與診斷………………………………. 3
1. 3. 1 肝癌早期發現與肝癌生物標記……………………. 4
1. 4 蛋白質體學的興起…………………………………. 6
1. 4. 1 蛋白質體學與肝癌生物標記………………………. 7
1. 4. 2 尋找腫瘤標記之新策略-組織間液………………… 7
1. 5 論文研究目的………………………………………. 9
第二章 實驗材料與方法
2. 1 實驗樣品收集………………………………………. 10
2. 1. 1 肝癌組織取得與保存………………………………. 10
2. 1. 2 肝癌組織間液製備…………………………………. 10
2. 1. 3 肝癌組織均質………………………………………. 11
2. 1. 4 臨床病患血清分離…………………………………. 11
2. 2 實驗方法……………………………………………. 12
2. 2. 1 樣品前處理…………………………………………. 12
2. 2. 2 二維電泳……………………………………………. 12
2. 2. 3 螢光差異蛋白表達分析系統………………………. 14
2. 2. 3. 1 樣品製備……………………………………………. 14
2. 2. 3. 2 CyDye製備與蛋白質標記………………………….. 14
2. 2. 3. 3 DIGE二維電泳……………………………………… 15
2. 2. 4 電泳膠影像掃描及分析……………………………. 16
2. 2. 5 膠體內蛋白質酵素水解……………………………. 16
2. 2 .6 MALDI-TOF質譜分析與蛋白質鑑定…………….. 17
2. 2. 7 西方點墨法…………………………………………. 18
2. 2. 8 In-house酵素連結免疫吸光法…………………….. 19
2. 2. 9 統計分析與蛋白質預測伺服器……………………. 21
第三章
3. 1 肝臟TIF/NIF以及TL/NL中蛋白質表現概況….... 23
3. 2 表現量相異之蛋白質分析與鑑定…………………. 23
3. 3 肝癌生物標記候選蛋白質在組織間液的表現……. 25
3. 4 血清中CyPA、ERp57以及RBP4含量檢測 ….… 26
3. 5 Cyclophilin A之ROC曲線………………………... 26
第四章 討論 …………………………………………………… 28
參考文獻…………………………………………………………… 37

圖表目錄
圖一、組織間液與組織均質液之蛋白質二維電泳……………… 45
圖二、組織間液以及組織均質液中表現量相異蛋白質以及組織間液蛋白質鑑定圖譜 ……………………………………………… 50
圖三、五個候選蛋白質在九對組織間液中表現局部圖與鑑定結果 52
圖四、候選蛋白質在組織間液的表現量………………………… 62
圖五、病患血清中蛋白質含量分析……………………………… 64
圖六、病患血清CyPA與AFP之ROC curve…………………… 66
表一、組織間液蛋白質改變量與鑑定資料表…………………… 68
表二、組織均質液蛋白質鑑定資料表…………………………… 77
表三、組織間液與均質液蛋白質細胞位置預測表……………… 93
表四、組織間液與均質液蛋白質生物功能預測表 ……………… 94
表五、組織間液與均質液蛋白質屬於典型或非典型分泌蛋白質預測表
…………………………………………………………… 95
表六、選為初步分析之肝癌生物標記候選蛋白質資料表 ……… 96
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