大腸直腸癌為常見的消化道惡性腫瘤，對於局部進行期的中低位直腸癌的臨床醫療方式，經常事先以手術前放射治療合併化學治療(Concurrent chemoradiotherapy CCRT)對病患腫瘤組織進行處理。但是接受CCRT的病患，卻常因個體間的差異性而產生不同的治療效果，因此，預先評估病患癌組織對於CCRT的敏感差異性，進一步制定個人化的CCRT療程，便成了這類患者治療計畫設計的重要參考指標。此研究主要利用本實驗室先前成功開發的專利平台-加權酵素型晶片操作平台（WEnCA），來分析大腸直腸癌患者在接受CCRT後，患者腫瘤細胞中與CCRT療效相關候選基因群的表現，並將候選基因表現結果與患者實際的臨床療效作統計分析，鑑別可作為預測病患對於CCRT是否具有感受性的分子標記。研究方法首先選擇與CCRT療效相關的候選基因共27個，包括來自本研究團隊先前的研究成果，以及近幾年來，國內外學者所發表透過比較放射敏感性及放射抗性細胞而篩選出與放射治療感受性有關之基因群，其中17個與腫瘤缺氧相關、6個與腫瘤惡性化相關以及4個基因先前已經臨床測試證實為放射線敏感性基因。接著，分別設計此27個候選基因的寡核甘酸片段，經過濃度的連續稀釋，找出每一基因最佳在晶片上的點漬濃度，繼而設計矩陣排列建構測試晶片，再利用WEnCA操作平台一一分析臨床所收集的66位大腸直腸癌病患血液檢體中CCRT療效候選基因的表現情況，並將實驗數據與臨床治療結果進行生物統計分析。在實驗結果中，我們發現27個候選基因中，有6個基因 PTMA(P =0.045)、GLUT1(P =0.001)、 BNIP3L(P =0.047)、 KCTD11(P =0.038)
、 PFKFB3(P =0.045)、 DDB2(P =0.045) 在大腸直腸癌病患有效及無效兩個族群間的差異表現，具有統計上的意義。其中，PTMA、GLUT1及PFKFB3三個基因在CCRT無效的族群中過度表現，而BNIP3L、KCTD11和DDB2三個基因則是在CCRT有效的族群中呈現過度表現。另外，也證實DDB2的過度表現與癌症患者的淋巴浸潤有關。進一步，我們將以本研究結果做為基礎，結合多種加權方式，並透過不同基因群的組合，確認最佳的候選基因群以及個別基因最適合的加權值，以建置臨床上實用的「化學治療合併放射治療療效預測晶片」，提供臨床醫師準確的療效預測結果，作為治療規劃的重要參考依據。

Colorectal cancer (CRC) is a common gastrointestinal malignancy tumor. Combined with radiation and chemotherapy (Concurrent Chemoradiation Therapy; CCRT) before surgery is often used as a therapeutic strategy for tumor tissue of patients with low CRC. However, response to CCRT differs among individual tumors. Therefore, applying the individual cancerous tissue variability of radiation sensitivity to predict CCRT efficacy before treatment will become an important guideline for designing personalized treatment plans. In this study, we used Weighted Enzymatic Chip Array platform (WEnCA) to analyze candidate genes expression in CRC patient's tumor cells after treatment with CCRT. Then, we combined the results of candidate genes expression with clinical response of patients after CCRT for statistical analysis to validate the molecular markers that can predict clinical sensitivity for CCRT. Twenty-seven CCRT candidate genes were selected from our previous results and published references in which the radiation response-related genes were determined by analyzing differential gene expression between radiosensitive and radio-resistant cells. In 27 CCRT candidate genes, 17 genes are related to tumor hypoxia, 6 genes are related to tumor malignancy and 4 genes have been validated as radiation-sensitive genes by clinical testing. Subsequently, we designed oligonucleotides fragment from 27 candidate genes, and identified the used oligonucleotide concentration for each gene on chip by serial dilution. The peripheral blood samples of 66 CRC patients treated with preoperative CCRT were collected for analyzing 27 CCRT related genes expression in CRC patients. Then the association between CCRT related gene expression and clinical efficacy of CCRT was analyzed using statistical software. Our results show that among the 27 CCRT candidate
genes, PTMA (P =0.045), GLUT1 (P =0.001), BNIP3L (P =0.047), KCTD11 (P =0.038), PFKFB3 (P =0.045) and DDB2 (P =0.045) expression significantly differ between CCRT response group and non- response group. PTMA, GLUT1 and PFKFB3 are highly expressed in CCRT non-response group compared to there in CCRT response group, while BNIP3L, KCTD11 and DDB2 genes are highly expressed in CCRT response group compared to there in CCRT non-response group. We also confirmed that increased DDB2 expression is related to lymphoid infiltration of cancer patients. Based on the results of the present study, we will further design a variety of weighting methods for different combinations of genes to validate the best combination of candidate genes and the most appropriate weighted value for each gene. Establishment of the clinically useful CCRT efficacy prediction chip would improve the accuracy of prediction of CCRT efficacy, and provide a reference for improving CRC therapy.

論文審定書 i
論文公開授權書 ii
致謝 iii
中文摘要 iv
Abstract vi
目次 viii
圖次 xi
表次 xii
第一章、緒論 1
1.1 研究背景及文獻回顧 1
1.1.1 大腸直腸癌的發生率 1
1.1.2 大腸直腸癌的診斷 1
1.2 病史 1
1.2.1 大腸直腸癌的症狀 2
1.2.2 糞便潛血檢查(Fecal occult blood test, FOBT) 2
1.2.3 理學指診檢查(Digital rectal examination) 2
1.2.4 乙狀結腸鏡檢查(Flexible sigmoidoscopy) 3
1.2.5 全大腸鏡檢查(Colonoscopy) 3
1.2.6 大腸鋇劑攝影(Bariun enema) 3
1.2.7 實驗室檢查 3
1.2.8 病理學檢查 4
1.3 大腸直腸癌的治療 4
1.3.1 手術切除為主要治療方式 4
1.3.2 輔助性治療 4
1.3.3 手術前放射治療 5
1.3.4 化學治療 5
1.3.5 合併放射與化學藥物輔助性治療 5
1.4 影響病患對於化學治療合併放射治療療效之因素 6
1.4.1 腫瘤的異型性 6
1.4.2 腫瘤之基因表現的差異 7
1.5 個人化醫療 7
1.6 CCRT相關分子標記研究之文獻回顧 8
1.7 腫瘤分子標記之偵測 9
1.8 研究目的 10
1.9 研究流程 11
1.10 研究流程說明 12
第二章、實驗材料與方法 13
2.1 CCRT療效檢測晶片之設計與製備 13
2.1.1 基因群篩選與寡核苷酸設計 13
2.1.2 基因晶片製作流程 13
2.1.3 CCRT療效檢測晶片測試 13
2.1.4 CCRT療效檢測晶片核苷酸濃度調整 14
2.2 檢體收集 14
2.2.1 病患選擇 14
2.2.2 病患檢體收集 15
2.2.3 CCRT療程設計 15
2.2.4 療效判定標準 16
2.2.5 病理資料收集 16
2.2.6 對照組的血液檢體 16
2.3 WEnCA實驗操作流程 16
2.3.1 血液之核糖核酸萃取(RNA isolation) 17
2.3.2 由核糖核酸反轉錄為互補去氧核糖核酸(cDNA) 17
2.3.3 探針標記(Probe Synthesis) 17
2.3.4 晶片雜合反應(Pre-Hybridization and Hybridization) 17
2.3.5 清洗與呈色(Wash and Detection) 18
2.4 個別基因表現量校正 18
2.5 生物統計分析 19
第三章、結果 20
3.1 CCRT療效檢測晶片之製備 20
3.1.1 基因群篩選 20
3.1.2 寡核苷酸設計結果 20
3.1.3 CCRT療效檢測晶片核苷酸濃度調整結果 20
3.2 大腸直腸癌檢體分析結果 21
3.3 大腸直腸癌病患基因表現分析結果 21
3.4 六個CCRT相關基因表現與大腸直腸癌病患臨床病理表徵之關聯性 22
3.5 合併多重基因表現與大腸直腸癌病患CCRT療效之關聯性 22
第四章、討論 24
結論與展望 29
圖表 30
參考文獻 44

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