臺灣博碩士論文加值系統

中文總摘要
根據世界衛生組織的統計，肺癌是目前世界上最常見的腫瘤之一，並且為國人重要的癌症死亡原因。衛生署最近的研究調查顯示，在台灣平均每十萬人口中罹患肺癌的死亡率約為31人，其中女性與男性肺癌的發生率分別佔惡性腫瘤的第一位與第二位，儘管近年來診斷與手術治療方法不斷改進，此發生率卻持續地增加中。由於腫瘤本身的生物學特性，所以大部分的病患無法在腫瘤初期即被發現，除非腫瘤嚴重侵犯到鄰近器官引起續發狀況，否則並不易被發現。因此尋找一種有效且可信度高的腫瘤分子標記對於這些病患的臨床追蹤是迫切需要的，不但可以早期偵測肺癌，甚至可作為監控肺癌的分子診斷標記，更可以因此而降低台灣肺癌的死亡率。近年來隨著分子生物學的進步，對於癌症發生及轉移的分子機制相關基因的變化逐漸被闡明，因此臨床上利用各種致癌基因與抑癌基因，進行癌症患者的預後評估變得相當有意義。為了可以更準確的早期診斷肺癌，首先必須確認與肺癌癌化有關的基因，釐清相關之腫瘤抑制基因與致癌基因在肺癌形成過程中所扮演的角色，並藉由了解參與癌症形成之基因，進一步發展可進行癌症診斷或治療的分子技術。因此為了更深入了解台灣地區肺癌形成過程所參與變異之基因種類，吾人首先收集佔所有肺癌75%之非小細胞肺癌 (NSCLC) 患者之組織與血液，分析腫瘤抑制基因p53及致癌基因K-ras。分析p53基因發現61位NSCLC中有27位突變，共有34個位置圖變點，其突變率為44% (27/61) ，大多屬於框架轉移突變 (frameshift) 且多為新的突變熱點 (hot spot)。另外，K-ras致癌基因在76位非小細胞肺癌中有28位病人有突變，其突變率為36.8% (28/76)，點突變 (point mutation) 位置主要集中在密碼12 (21/28; 75.0%) 與13 (5/28; 17.8%)，突變也好發在有抽菸的非小細胞肺癌病患 (51.4% vs. 24.4%, p＜0.05) 。由上述結果，吾人希望這些屬於國人特有的基因表現頻率與圖譜可進一步作為研發肺癌分子標記的基礎。延續第一個計畫，利用高效能 (high throughput) 之微矩陣列分析與生物資訊軟體 (Spotfire及GeneSpring) 之運用，找出腫瘤組織與正常組織中的差異性表現基因。嘗試分別由腺癌、鱗狀細胞癌與大細胞癌等，3對非小細胞肺癌腫瘤組織與相對的鄰近正常肺組織檢體進行微矩陣列分析，從中找出屬於台灣非小細胞肺癌特異性標的基因作為研發完整的癌症基因診斷與治療標的。從微矩陣列的實驗結果中得知有3625個基因具差異性表現情形，1720個基因為升調節 (up regulation；tissue/normal ratio ≥ 2)；1905個基因為降調節 (down regulation；tissue/normal ratio ≤ 0.5) 。由生物資訊分析基因表現路徑發現，從1720個升調節基因中有95個基因參與Cytokine-cytokine receptor interaction、84個基因參與Focal adhesion與72個基因參與MAPK signaling pathway等三大路徑 (p≤0.05)。詳細探討此三大路徑內基因表現情形及其相互關係後得知，其中Focal adhesion為一調控細胞功能與腫瘤起始相關作用的核心路徑，且具有與其他路徑相互關聯的基因群。同時，吾人也將此部分研究中8個顯著的升調節基因(up regulation；tissue/normal ratio ≥ 20) 的8個非小細胞肺癌特異性基因，與4個由文獻探討後發現與肺癌致癌過程相關且具有診斷潛力的基因標的，整合此12個特異性較佳的基因共同作為肺癌輔助診斷的基因標的，進一步將測試這些基因標的在肺癌患者體內之表現情形。首先，選擇定量PCR (qRT-PCR) 這一種靈敏度相當高的技術為基礎，比較本實驗室於先前建立可以同時偵測多項基因表現的尼龍膜陣列技術平台，結果發現此兩種方法有相當一致的結果 (r＝0.921)。再者，經由承受操作曲線特性 (ROC curve) 確認此12個有明顯升調節的基因群，可整合作為診斷肺癌之肺癌特異性的標的基因群，作為輔助肺癌診斷的基因標的。統合非小細胞肺癌病患臨床病理報告分析，結果發現hMTH1、SPD、HARP2、ITGA11、COL11A1與CK-19等六種訊息核醣核酸 (mRNA) 分子標記的表現與病理分期有關，其中hMTH1、SPD、ITGA11與COL11A1之訊息核醣核酸分子標記又與淋巴結轉移及預後有關。此外，這套成本低、操作容易且效能高的癌症診斷法，呈色型的癌症診斷晶片，以化學呈色的方式取代傳統微矩陣列分析的螢光呈色、以尼龍膜取代傳統的玻璃晶片，不但大幅降低儀器及試劑成本更突破多重技術門檻。並且整個基因陣列尼龍膜片的靈敏度 (sensitivity) 為89%及特異性 (specificity) 為84%顯示具有相當好之輔助診斷價值。同時吾人也清楚發現利用多個癌症分子標記的組合 (訊息核醣核酸、去氧核醣核酸 (DNA)) 用在非小細胞肺癌上，可顯著提高臨床患者的早期診出率及臨床診斷的正確性，更遠較於傳統所使用的腫瘤標記為佳。综合這些有意義的肺癌基因群，吾人深信這些分子標記，將有助於臨床醫師迅速並有效地早期診斷肺癌，以期能更早普及化的預防與早期治療，並提升肺癌病患的存活率與治癒率。

Abstract
Lung cancer in Taiwan has been the leading and second-leading cause of cancer deaths in women and men, respectively, since 1985. Although there have been tremendous improvements in diagnostic methods and surgical treatments, the incidence of lung cancer is still on the rise. Due to the biological characteristics of the tumor itself, most patients are unaware of the malignant tumor during the early-stage. However, a series of symptoms occur when the malignant tumor severely attacks neighboring organs. Therefore, the search for a highly effective and reliable tumor molecular marker for clinical applications, such as early lung cancer detection and cancer surveillance, is important and urgent due to the nature of the tumor. Furthermore, the impact of a tumor molecular marker may decrease the lung cancer mortality rate in Taiwan. Over the past few years, the development of molecular biology has gradually defined the changes in gene expression that are associated with carcinogensis and metastasis. These developments enable meaningful clinical use of the various oncogenes and tumor suppressor genes for the prognosis of cancer. In order to detect lung cancer early, the first step is to recognize the genes that are associated with carcinogenesis and clarify the oncogenes and tumor suppressor genes that participate in the tumorgenesis. We first collect of blood and tissue from patients with NSCLC, the most frequent type of lung cancer. Our research mainly focuses on a tumor suppressor gene, p53 and an oncogene, K-ras which are both highly relevant in the carcinogenesis of NSCLC. We investigate the mutations of the two genes in lung cancer tissues within Taiwanese lung cancer patients. Our results have shown that among 61 NSCLC patients that have p53 mutation, 34 mutations of the p53 gene were found in 27 cases with a mutation rate of 44% (27/61). Most mutations are frameshift mutations and are mostly new mutation hot spots. On the other hand, there are 28 patients out of 76 Taiwanese NSCLC patients with mutations in the K-ras oncogene, which corresponds to a mutation frequency of 36.8% (28/76). Most of the K-ras mutations are missense mutations at codon 12 (17/23; 73.9%) and 13 (5/23; 21.7%). The K-ras mutations were more frequently found in smokers than in non-smokers (51.4% vs. 24.4%, p＜0.05). According to the above results, we expect that the mutation frequency and patterns of particular genes, specifically for Taiwanese citizens, can be used as the basis toward the development of biomarkers for lung cancer. Following the attempt to search for novel candidate genes specifically for Taiwanese citizens, we combined high-throughput technologies by utilizing microarray systems and bioinformatic software (Spotfire ＆ GeneSpring) to identify the differential gene expression within tumor tissues. Initially, we used a microarray to analyze the differential expression of 3 NSCLC paired tissue samples, seeking for biomarkers specifically for NSCLC (in Taiwan) that can be used in the development of cancer diagnosis and target therapy. From the result of the microarray, we discovered 1720 up-regulated genes (tissue/normal ratio ≥ 2) and 1905 down-regulated genes (tissue/normal ratio ≤ 0.5). After analyzing the up-regulated genes from the microarray data, we discovered that the expressed genes mostly participated in three pathways: Cytokine-cytokine receptor interaction, Focal adhesion and MAPK signaling pathway. Each pathway contains 95, 84, and 72 genes, respectively. After investigating the activations and the interactions of the genes within these three pathways, the Focal adhesion pathway was found to be the main pathway which regulates the function of the cell and the carcinogenesis of cancer. It also has a group of genes that interact with each other. As for developing specific biomarkers for lung cancer diagnosis, we combined 8 significantly up-regulated genes (tissue/normal ratio ≥ 20) expressed in NSCLC and 4 genes which can possibly be the most promising diagnosis markers and have already been identified to participate in the carcinogenesis of lung cancer. Primarily, this study compares the quantified PCR with the membrane array, a technique which can detect multiple gene expressions at the same time, to elucidate any correlation between the two methods. The result indicated that these two methods were highly correlated (r=0.921). Next, by the ROC curve analysis, this study confirmed that the 12 significantly up-regulated genes can be used as specific mRNA markers for the future diagnosis of lung cancer. Upon the clinicopathological data of the patients being further analyzed with the 12 biomarkers, we discovered the expression of 6 mRNA markers－hMTH1, SPD, HARP2, ITGA11, COL11A1 and CK-19. These markers were associated with clinicopathological stages while the hMTH1, SPD, ITGA11 and COL11A1 makers were associated with the prognosis and lymphatic metastasis. In addition, the cancer diagnostic chip－membrane array, which was established in our lab, has the advantage of low cost, easy operation and high efficiency. Under such conditions, the sensitivity and specificity of the mRNA markers panel for NSCLC were 89% and 84%, respectively. Compared to traditionally-used tumor markers, this research clearly indicates that the combination of multiple gene markers for the diagnosis of NSCLC may significantly increase the accuracy and the efficacy for early detection of lung cancer. With the practice of lung cancer-specific mRNA markers, we believe this could be helpful for physicians to detect lung cancer more quickly and effectively. Earlier detection will lead to faster treatment and improve the prognosis and survival rate of lung cancer patients.

目 錄

中文總摘要 4
Abstract 8
圖與表目錄 12
第一章 研究背景與文獻回顧 14
一、肺癌為惡性腫瘤死亡原因之第一位 15
二、肺癌的病理組織型態分類與臨床診斷16
三、肺癌可能發生的原因 17
四、基因體學及其生物技術之應用 19
五、肺癌早期診斷之理論 20
六、研究概述 21
縮寫表 23
第二章 p53基因在人類非小細胞肺癌突變情形分析 25
前言 26
材料與方法 29
結果 36
討論 38
流程圖 (一) 41


第三章 K-ras基因在人類非小細胞肺癌突變情形分析 51
前言 52
材料與方法 54
結果 56
討論 57
第四章 非小細胞肺癌本土性差異表現基因之分析 63
前言 64
材料與方法 66
結果 68
討論 69
流程圖 (二) 71
第五章 非小細胞肺癌在生物反應路徑中明顯表現的基因 77
前言 78
材料與方法 79
結果 80
討論 82
流程圖 (三) 84


第六章 非小細胞肺癌基因診斷晶片之評估 89
前言 90
材料與方法 93
結果 96
討論 99
流程圖 (四) 103
第七章 總結論 112
第八章 參考文獻 116
附件 已刊登之論文 132

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葉昌偉, and 謝昌煥. 基因晶片簡介與分析及應用軟體介紹(上).