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研究生：	黃永敬 Huang, Yung-jing
論文名稱：	利用即時聚合酶連鎖反應技術分析人類大腸直腸癌組織中表皮生長因子接受器及其受質基因之表現 Analysis of the Expression Level of Epidermal Growth Factor Receptor and Its Ligands in Human Colorectal Cancer by Real Time PCR
指導教授：	蕭世裕 Shaw, Shyh-Yu
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2009
畢業學年度：	97
語文別：	中文
論文頁數：	104
中文關鍵詞：	表皮生長因子受器、大腸直腸癌、表皮生長因子受質、即時聚合酶連鎖反應
外文關鍵詞：	Real-time PCR, Epidermal Growth Factor Receptor, Epidermal Growth Factor Receptor ligands, Colorectal Cancer
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表皮生長因子受器 (Epidermal growth factor receptor, EGFR) 在調控上皮細胞的增生、分化及存活上扮演著重要角色，EGFR 訊息傳遞路徑失去調控會造成細胞不正常的增生及分化，進而造成各種癌症的產生。大腸直腸癌 (Colorectal cancer，CRC) 被認為是由於 EGFR 蛋白質或其 mRNA 大量表現所造成，臨床研究也顯示有25至75% 病患之大腸癌組織呈現 EGFR過量表現的情形。目前最普遍用來偵測EGFR表現的方法為免疫組織化學染色法(Immunohistochemistry staining，IHC)，臨床上是以其結果作為對病患是否施以標靶治療之依據，但是有越來越多的臨床報告指出IHC的結果與標靶治療的反應並無絕對的相關性。此外，也有報告指出部分 CRC 組織之EGFR 基因複製數目（Copy number）有增加的現象，因此利用螢光原位雜交(Fluorescence in situ hybridization，FISH）來偵測 EGFR 基因複製數目的方法也被用於評估 CRC 之診斷與治療。然而，許多最近的研究顯示僅有少數的 CRC 組織之EGFR 基因複製數目有增加的現象。因此，截至目前為止尚未有一個明確的分子標記可作為 CRC臨床治療的指標。本研究的目的是利用即時聚合酶連鎖反應（Real-time PCR）來測定大腸癌組織的EGFR 與其各種ligands之mRNA的表現量，與病歷資料做統計，希望能找出CRC的特定分子標記以提供臨床治療的參考。目前我們已分析85位 CRC 病患的 EGFR 及 EGFR ligands (包括Epiregulin、Amphiregulin、Transforming Growth Factor-α、Epidermal Growth Factor、Heparin-binding EGF-like growth factor、Betacellulin)之 mRNA 在正常組織與癌組織的表現，發現EGFR mRNA在大腸直腸癌組織相對表現量(10.75 ± 5.38)比癌組織周圍正常組織(12.72 ± 4.73)有較低的趨勢，具統計意義( p = 0.007)，但存活分析發現沒有顯著差異( p > 0.05)。而EGFR ligands中發現EREG、AREG mRNA在癌組織(100.73 ± 147.41、10.81 ± 13.00)比癌組織周圍正常組織(17.46 ± 42.81、5.27 ± 5.86)中皆有高顯著表現( p<0.0001)，由統計分析和存活分析結果發現EREG、AREG 之mRNA相對表現量高病人存活比例較高( p = 0.024、0.02)，而且存活時間較久( p = 0.04、p = 0.04)。依本實驗之結果推斷EREG、AREG mRNA在大腸直腸癌中相對表現量可以當作臨床診斷的指標，而且EREG與AREG之高表現量可能降低病人之死亡率。

Epidermal growth factor receptor (EGFR) plays a pivotal role in regulating epithelial proliferation, differentiation and survival. Aberrant regulation of EGFR signaling has been implicated in the development and progression of several different solid tumors. Colorectal cancer (CRC) is considered a tumor showing EGFR overexpression alternatively in protein or mRNA levels. Clinical studies have proven that 25~ 75% of CRC patients have shown high EGFR expression. Now the most popular method for EGFR determination is immunohistochemistry staining (IHC) that was currently used as a criterion for EGFR target therapy. More and more reports, however, have suggested that there is no absolutely correlation between IHC-positive and target therapy response. Recently, the increased EGFR gene copy number has also been found in some CRC case. Fluorescence in situ hybridization (FISH) method to detect the EGFR gene copy number was therefore developed for CRC diagnosis and therapy. But there are also many reports stated that only small minority case with increased CRC gene copy number. Up to now, there is not a single molecular marker could be used as a certainly criterion in CRC therapy. The aim of this study is to shed more light on EGFR expression in CRC, evaluating the expression of EGFR and EGFR ligands using real-time PCR method and statistical analyses with case history. We have already analyzed that 85 CRC patients mRNA of EGFR and EGFR ligands in normal and cancer tissues. Finding EGFR mRNA has lower expression level in the cancer tissue (10.75 ± 5.38) as compared to normal tissues (12.72 ± 4.73), according to the statistics ( p = 0.007). But survival analysis has not shown the difference ( p > 0.05). The result was similar with many other reports that implicated the failure of predicting EGFR mRNA expression as a CRC therapy indicator. In contrast, two of EGFR ligands’, EREG and AREG, mRNA ( p < 0.0001) expressed high level in cancer tissues (100.73 ± 147.41 and 10.81 ± 13.00) as compared to normal tissues(17.46 ± 42.81 and 5.27 ± 5.86). Statistical analysis and survival analysis showed that the high expression level of EREG and AREG mRNA is associated with the rate of patient survival (p = 0.024, 0.02) and the survival time ( p = 0.04, p = 0.04). According to this result, the high expression level of EREG and AREG mRNA in colorectal cancer, has the potential to be used as molecular markers in clinical diagnosis and it may associated with the survival rate of colorectal cancer patients.

目錄
中文摘要 ............................................................................................................ I
Abstract .......................................................................................................... III
目錄 .................................................................................................................. V
圖目錄 .......................................................................................................... VIII
表目錄 ............................................................................................................. XI
第一章 緒論 ................................................................................................... 1
第二章 文獻探討 ........................................................................................... 3
1. 大腸直腸癌 (Colorectal cancer，CRC) ............................................ 3
1.1 大腸直腸癌在台灣的現況 ........................................................... 3
1.2 大腸直腸癌的危險因子................................................................ 3
1.3 大腸直腸癌分期 ............................................................................ 6
1.4 大腸直腸癌治療 ............................................................................ 7
2. 表皮生長因子接受器（EGFR） ....................................................... 9
2.1 EGFR結構和功能 ........................................................................ 9
2.2 EGFR在癌症中重要性 .............................................................. 11
2.3 專一性抑制EGFR治療癌症細胞 ............................................. 12
3. Cetuximab .......................................................................................... 13
第三章 研究目的 ......................................................................................... 15
第四章 材料與方法 ..................................................................................... 17
一、 材料 ............................................................................................ 17
1. 樣品和試劑 ....................................................................................... 17
2. 引子（Primer）序列 ........................................................................ 20
二、 儀器 ............................................................................................ 20
三、 實驗方法 .................................................................................... 21
1. 組織來源 ........................................................................................... 21
2. 從組織中抽取全部的核糖核酸（RNA）....................................... 21
3. 測定核糖核酸（RNA）濃度 ........................................................... 23
4. 反轉錄聚合酶連鎖反應（RT-PCR）混合液配製和執行 ............. 24
5. 即時聚合酶連鎖反應（Real-time PCR）混合液配製和執行 ...... 25
6. 統計分析 ........................................................................................... 25
第五章 結果 ................................................................................................. 27
1. 利用Real- time PCR測定訊息核糖核酸（mRNA）表現 ............ 27
2. 訊息核糖核酸（mRNA）表現與病人臨床病歷資料統計分析 ... 33
2.1 描述性統計分析 .......................................................................... 33
2.2 獨立樣本T檢定 ......................................................................... 34
2.3 卡方分析 ...................................................................................... 36
3. 存活分析 ........................................................................................... 39
第六章 討論 ................................................................................................. 44
第七章 結論 ................................................................................................. 49
第八章 參考文獻 ......................................................................................... 50

圖目錄
圖一、表示EGFR家族四個成員和其受質。 ............................................ 55
圖二、說明EGFR訊號傳遞路徑下游對細胞與組織的影響。 ................ 56
圖三、表示全部85位病人中個別病人EGFR mRNA在正常組織和癌組織表現情形。 ................................................................................. 57
圖四、盒鬚圖為85位病人整體來看EGFR mRNA在正常組織和癌組織表現情形。 ..................................................................................... 58
圖五、表示全部85位病人中個別病人EREG mRNA在正常組織和癌組織表現情形。 ................................................................................. 59
圖六、盒鬚圖為85位病人整體來看EREG mRNA在正常組織和癌組織表現情形。 ..................................................................................... 60
圖七、表示全部85位病人中個別病人AREG mRNA在正常組織和癌組織表現情形。 ................................................................................. 61
圖八、盒鬚圖為85位病人整體來看AREG mRNA在正常組織和癌組織表現情形。 ..................................................................................... 62
圖九、表示全部85位病人中個別病人TGF-α mRNA在正常組織和癌組織表現情形。 ................................................................................. 63
圖十、盒鬚圖為85位病人整體來看TGF-α mRNA在正常組織和癌組織表現情形。 ..................................................................................... 64
圖十一、表示全部85位病人中個別病人EGF mRNA在正常組織和癌組織表現情形。 ................................................................................. 65
圖十二、盒鬚圖為85位病人整體來看EGF mRNA在正常組織和癌組織表現情形。 ..................................................................................... 66
圖十三、表示全部85位病人中個別病人HB mRNA在正常組織和癌組織表現情形。 ................................................................................. 67
圖十四、盒鬚圖為85位病人整體來看HB mRNA在正常組織和癌組織表現情形。 ..................................................................................... 68
圖十五、表示全部85位病人中個別病人BTC mRNA在正常組織和癌組織表現情形。 ................................................................................. 69
圖十六、盒鬚圖為85位病人整體來看BTC mRNA在正常組織和癌組織表現情形。 ..................................................................................... 70
圖十七、85位病人EGFR mRNA表現量以Kaplan-Meier統計來做存活分析。 ............................................................................................. 71
圖十八、85位病人EREG mRNA表現量以Kaplan-Meier統計來做存活分析。 ............................................................................................. 72
圖十九、85位病人AREG mRNA表現量以Kaplan-Meier統計來做存活分析。 ............................................................................................. 73
圖二十、85位病人TGF-αmRNA表現量以Kaplan-Meier統計來做存活分析。 ............................................................................................. 74
圖二十一、85位病人EGF mRNA表現量以Kaplan-Meier統計來做存活分析。 ............................................................................................. 75
圖二十二、85位病人HB mRNA表現量以Kaplan-Meier統計來做存活分析。 ................................................................................................. 76
圖二十三、85位病人BTC mRNA表現量以Kaplan-Meier統計來做存活分析。 ............................................................................................. 77

表目錄
表一、Dukes分期根據大腸直腸癌手術病理標本中癌細胞侵犯深度 ..... 78
表二、AJCC第六版美國聯合癌症委員會將大腸直腸癌分為0期至Ⅳ期79
表三、各種癌症中EGFR過量表現的比率 ................................................ 80
表四、引子（Primer）序列 .......................................................................... 81
表五、標準品核糖體RNA一系列濃度之配製方法 .................................. 82
表六、反轉錄聚合酶連鎖反應混合液之配製方法 ..................................... 83
表七、反轉錄聚合酶連鎖反應儀器之程式設定 ......................................... 84
表八、即時聚合酶連鎖反應混合液之配製方法 ......................................... 85
表九、即時聚合酶連鎖反應儀器之程式設定 ............................................. 86
表十、各基因訊息核醣核酸（mRNA）相對表現（N =85） ................... 87
表十一、大腸直腸癌病患基本資料（N =85） ........................................... 88
表十二、年齡與基因mRNA相對表現量分析表 ....................................... 89
表十三、性別與基因mRNA相對表現量分析表 ....................................... 90
表十四、腫瘤發生位置與基因mRNA相對表現量分析表 ....................... 91
表十五、大腸直腸癌病理分期與基因mRNA相對表現量分析表 ........... 92
表十六、病人存活狀況與基因mRNA相對表現量分析表 ....................... 93
表十七、病人復發狀況與基因mRNA相對表現量分析表 ....................... 94
表十八、只有接受手術治療與基因mRNA相對表現量分析表 ............... 95
表十九、化學治療與基因mRNA相對表現量分析表 ............................... 96
表二十、放射治療與基因mRNA相對表現量分析表 ............................... 97
表二十一、EGFR mRNA相對表現量高低與病人基本資料之分佈情況 . 98
表二十二、EREG mRNA相對表現量高低與病人基本資料之分佈情況 99
表二十三、AREG mRNA相對表現量高低與病人基本資料之分佈情況100
表二十四、TGF-αmRNA相對表現量高低與病人基本資料之分佈情況101
表二十五、EGF mRNA相對表現量高低與病人基本資料之分佈情況 . 102
表二十六、HB mRNA相對表現量高低與病人基本資料之分佈情況 ... 103
表二十七、BTC mRNA相對表現量高低與病人基本資料之分佈情況 . 104
                                    

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