臺灣博碩士論文加值系統

在台灣，根據衛福部去年公布的衛生統計年報顯示，大腸直腸癌死亡率高居所有癌症第三。世界衛生組織2014年全球癌症統計年報指出大腸直腸癌每年增加140萬名新病例，而罹患大腸直腸癌死亡則有70萬人。文獻指出Store-operated calcium channel (SOC)在乳癌、肺癌及大腸直腸癌扮演關鍵角色，其中Stromal interaction molecule (STIM1)為SOC調控機轉上的鈣離子感應器，STIM1的表現量會調控鈣離子的流量而影響癌細胞的爬行與轉移。先前已有研究闡述與大腸直腸癌相關之致癌基因和抑癌基因會受到DNA甲基化所調控，因此本研究希望藉由分析大腸直腸癌患者之STIM1啟動子上的轉錄因子結合位之甲基化程度，探討STIM1基因甲基化與大腸直腸癌臨床表徵之間的關聯性，作為未來臨床診斷之參考。本研究共納入108位受過根治性手術和FOLFOX治療之大腸直腸癌患者，並在STIM1啟動子上選定被證實與致癌機轉具有高度相關之NF-κB、E2F1與CREB1結合位進行分析。經pyrosequencing分析上述轉錄因子結合位的CpG甲基化程度合併臨床資料統計分析，結果發現位於NF-κB結合位之CpG高度甲基化與大腸直腸癌遠端轉移之相關性有顯著意義(p = 0.008)，NF-κB結合位高度甲基化患者之無疾病存活率亦顯著低於低度甲基化之病人(p = 0.009)。此外在E2F1與CREB1結合位之DNA甲基化與癌細胞分化程度達到顯著統計意義。本研究證實NF-κB結合位的高甲基化組與大腸直腸癌術後遠端轉移之情形有正向相關，且造成病人之無疾病存活期下降。做為首篇針對大腸直腸癌化學治療之STIM1 DNA甲基化的研究，我們期望本研究之結果能促使未來臨床上將DNA甲基化作為大腸直腸癌的診斷指標之一，提供癌症病患早期診斷與預防。

In Taiwan, according to the annual report of death statistics from Ministry of Health and Welfare last year, colorectal cancer (CRC) was the third leading cause of death among whole cancers. According to the report of GLOBOCAN 2014, there are 1.4 million new cases and 70 million deaths as a result of CRC each year. Literature has demonstrated an important role of store-operated calcium channel (SOC) in breast cancer, lung cancer and colorectal cancer. Moreover, stromal interaction molecule (STIM1) has been known to serve as a calcium sensor in the SOC-mediated pathway and regulate calcium influx which influences migration and metastasis of cancer cells. Previous studies have indicated that CRC-related oncogenes and tumor suppressor genes were regulated by DNA methylation. The aim of the study was to identify the association between STIM1 methylation and CRC clinical features through analyzing DNA methylation of transcription factor binding sites in the STIM1 promoter region. In the study, we enrolled a total of 108 CRC patients undergoing radical surgery and FOLFOX chemotherapy and selected binding sites of NF-κB, E2F1 and CREB1 in the STIM1 promoter which have been proven to be highly associated with carcinogenesis. Using the pyrosequencing method to analyze methylation of CpG islands in target transcript factor binding sites, we found high methylated NF-κB binding sites being associated with post -operation distant metastasis in CRC patients (p = 0.008). Patients with high methylated NF-κB showed lower disease free survival rate than those with low methylation (p = 0.009). Additionally, our result revealed that DNA methylation of CREB1 and E2F1 binding sites were significantly associated with histology of CRC. The study indicated that highly methylated NF-κB binding site was positively correlated with post-surgical distant metastasis decreased CRC patients’ disease free survival rate. We expected the results of the study able to promote the DNA methylation as a clinical diagnostic biomarker of CRC, providing early diagnosis and precaution for cancer patients.

中文摘要 I
英文摘要 III
致謝 V
目錄 VII
表目錄 IX
圖目錄 X
壹、研究背景介紹 1
一、鈣池調控鈣離子通道 1
二、大腸直腸癌(Colorectal cancer/CRC) 8
三、DNA甲基化 14
貳、研究動機 16
参、材料與方法 17
一、細胞培養(cell culture) 17
二、短暫性DNA轉染 (Transient transfection) 19
三、組織蛋白質萃取 20
四、酵素連結免疫吸附法 21
(Enzyme-Linked lmmunosorbent Assay , ELISA) 21
五、鈣離子訊息測定 22
六、蛋白質萃取 24
七、西方墨點法 (Western blotting) 26
八、研究對象 29
九、倫理聲明 29
十、臨床資料蒐集 30
十一、組織DNA萃取 31
十二、重亞硫酸鹽轉化(Bisulfite conversion) 33
十三、甲基化核酸引子設計 35
十四、甲基化聚合酶連鎖反應 (Pyromark PCR) 38
十五、DNA洋菜膠電泳 (Agarose gel electrophoresis) 39
十六、焦磷酸測序(Pyrosequencing) 40
十七、測序結果判讀 43
十八、統計方法 44
肆、實驗結果 45
伍、結論 55
柒、參考文獻 66
捌、附錄 74

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