臺灣博碩士論文加值系統

　　近年來，隨著環境及飲食習慣的改變，根據行政院衛生福利部國民健康局公布2011年台灣地區診斷為直腸結腸癌的個案有14040位，已躍為十大癌症發生率的第一名。儘管醫療的進步，直腸結腸癌的分子機轉仍尚未完全釐清。至今直腸結腸癌形成的原因是因為基因(genetic)和表顯遺傳(epigenetic) 修飾的改變所造成。而本研究目的找出大腸直腸癌具過度甲基化之基因BEND5，並深入探討BEND5 mRNA及啟 子甲基化之情形，於癌細胞中扮演的功能與角色之影響的致癌機轉，以助於了解BEND5於癌症變異情形及形成原因。
　　本研究將從醫院取得的大腸直腸癌手術後臨床檢體，抽取DNA後做bisulfite convert DNA進行甲基化分析，利用Illumina Human Methylation450觀察到BEND5 promoter上有高度甲基化的情形;進一步以Real-time methylation specific PCR (qMSP) 偵測發現有82.35% (70/85位) BEND5 promoter上具高度甲基化的情形，特別是在有局部淋巴結轉移的病人癌組織中95.2% (20/21) (p= 0.030)。再利用Real-time PCR分析，相較於正常組織71% (24/34位) 病人的病灶BEND5 mRNA都是低表達。為了瞭解BEND5在癌細胞中所具有的功能，藉由大腸癌細胞株實驗將BEND5大量表現於大腸癌細胞中，可以看到細胞增殖的數目相對的比控制組少。若同時將大量表現BEND5的細胞剔除 BEND5 mRNA於細胞中的表現，其細胞增殖的數目回復到正常狀態。本研究推測BEND5在大腸癌形成可能扮演抑癌基因的角色。

Colorectal cancer (CRC) is the most frequently diagnosed cancer in Taiwan. Colorectal cancer occurrence rates have been increasing in recent years. It was the third leading cause of cancer death on 2011. DNA methylation is common abnormality in colorectal cancer (CRC). Aberrant promoter hypermethylation of TSG-associated CpG islands can lead to transcriptional silencing and result in tumorigenesis. DNA methylation is catalyzed by enzymes known as DNA methyltransferases. We applied Illumina human methylation 450K to identify loci hypermethylation in 26 primary CRCs relative to non-neoplastic colorectal tissue. A candidate tumor suppressor gene, BEND5 showed 82.35% (70 of 85) hypermethylation in colorectal tumors compared to matched normal colorectal tissues, especially in patients with metastasis in regional lymph nodes 95.2% (20/21) (p = 0.030). This CRC-associated hypermathylation events were evaluated by quantitative methylation specific real-time PCR (qMSP-PCR). Using real-time PCR found 71% (24 of 34) BEND5 mRNA expression was down-regulation in CRCs compared to matched normal colorectal tissues. Transient transfection of BEND5 and/or si-BEND5 found BEND5 could inhibit colon cancer cell proliferation. In addition, BEND5 was expressed in nucleus by detecting immunofluorescence when BEND5 was transfected into cell line. In conclusion, BEND5 alteration may play an important role in colorectal cancer. The predominana mechanisms of BEND5 inactivation were BEND5 promoter hypermathylation. The present study suggested that the alteration of BEND5 may be involved in tumor development. We thought BEND5 is a candidate tumor suppressor gene in colorectal cance.

目錄 I
表目錄 II
圖目錄 III
中文摘要 IV
Abstract VI
壹、緒論 1
第一節　大腸直腸癌簡介 1
第二節　基因與表顯遺傳的調控 2
第三節　DNA甲基化與致癌機制之關聯 6
第四節　BEND5基本介紹及與癌症之關係 8
貳、研究目標 11
參、實驗材料與方法 12
第一節　研究材料與試劑 12
第二節　儀器與設備 16
第三節　實驗方法 18
肆、實驗結果 27
第一節　BEND5基因啟動子過度甲基化情形 27
第二節　大腸癌病患BEND5之甲基化情形與病歷資料相關性 28
第三節　大腸癌患者BEND5 mRNA於病灶呈現低表達 29
第四節　轉染BEND5入DLD-1細胞對於細胞型態及生長速率之分
析 29
第五節　BEND5造成細胞週期分布的改變 30
第六節　細胞以si-DNMT1分析DNMT1對於BEND5的DNA甲基化及
基因表現 30
伍、結論與建議 32
附表 37
附圖 39
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行政院衛生福利部國民健康署，民國100年癌症登記報告，2014。