臺灣博碩士論文加值系統

肝癌（Hepatocellular carcinoma，簡稱HCC）在全世界是個高發生率與高死亡率的癌症。許多研究已指出，異常的DNA甲基化模式在肝癌的形成中扮演著重要的角色。實驗室先前利用Methylation array篩選出許多在肝癌檢體中DNA甲基化之模式異常的候選基因；並且，使用甲基化專一性聚合酶鏈鎖反應（MS-PCR）與亞硫酸鹽定序(Bisulfite sequencing)驗證Methylation array的結果。雖然MS-PCR與bisulfite sequencing為分析DNA甲基化的標準方法，但這兩種方法只能提供定性或半定量的分析結果。Pyrosequencing為近年來新發展的定序技術，可以定性與定量分析DNA甲基化程度。因此，本論文的目標是建立Pyrosequencing平台用以進行DNA甲基化分析。
本論文先以控制組基因GLOXD1驗證Pyrosequencing平台分析之結果與MS-PCR和Bisulfite sequencing分析之結果一致。再以Pyrosequencing平台驗證候選基因在肝癌細胞與肝癌組織檢體裡存在有高度甲基化現象（ALDH1A與CALCA）或低度甲基化現象（DNMT3A1與DNMT3A2）。研究結果顯示我們成功的建立Pyrosequencing平台，並且證明它是個好的實驗平台，可應用於驗證候選基因的DNA甲基化程度。

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and is a leading cause of cancer death. Differential DNA methylation pattern plays an important role in the development of HCC. Previously, aberrant methylated candidate genes in HCC were selected using methylation array and validated by methylation-specific PCR (MS-PCR) and bisulfite sequencing. Although MS-PCR and bisulfite sequencing are the standard methods methylation analysis, they only provide qualitative or semi-quantitative data. Recently, pyrosequencing, a sequence-based detection technology, has been shown that it can accurately provide quantitative data for DNA mehtylation. The aim of this study is to establish a pyrosequencing platform for DNA methylation analysis.
In this study, we first established the pyrosequencing platform using a known gene GLOXD1, which is a hypermethylated gene. We then tested the feasibility of this platform in DNA methylation analysis compared with other methods, such as MS-PCR and bisulfite sequencing. Aberrant methylated candidate genes, including ALDH1A2, CALCA, DNMT3A1 and DNMT3A2, were validated as hypermethylated or hypomethylated in HCC cell lines and tissues by pyrosequencing. These data demonstrate that pyrosequencing platform is established and it is a good strategy to validate methylation levels of candidate genes.

第一章、緒論......................................1
第一節、肝癌的風險因子與致病機轉.....................1
第二節、DNA甲基化作用與癌症形成之相關性...............2
第三節、DNA甲基化轉移酶與癌症形成之相關性.............3
第四節、分析DNA甲基化的方法及限制....................4
第五節、研究動機..................................8
第二章、材料與方法................................9
第一節、分析候選基因在細胞株與組織檢體之DNA甲基化程度...9
壹、細胞株與組織檢體來源...........................9
貳、細胞株DNA萃取................................9
参、組織檢體DNA萃取..............................10
肆、亞硫酸鹽轉換（Bisulfite conversion)...........11
伍、焦磷酸測序（Pyrosequencing）Primer設計..........12
陸、聚合酶鏈鎖反應（Polymerase chain reaction）.....12
柒、巢式聚合酶鏈鎖反應（Nested-PCR）................14
捌、焦磷酸測序（Pyrosequencing）...................15
第三章、結果......................................17
第一節、建立Pyrosequencing平台.....................17
壹、細胞株裡GLOXD1啟動子區域甲基化程度................17
貳、組織檢體裡GLOXD1啟動子區域甲基化程度..............18
第二節、以Pyrosequencing分析候選基因的DNA甲基化程度...19
壹、候選基因ALDH1A2啟動子區域甲基化程度之分析..........19
貳、候選基因CALCA啟動子區域甲基化程度之分析............21
参、候選基因DNMT3A1啟動子區域甲基化程度之分析..........22
肆、候選基因DNMT3A2啟動子區域甲基化程度之分析..........24
第四章、討論.......................................26
第五章、結論...................................... 33
第六章、參考文獻....................................80

表一、候選基因之Pyrosequencing primer...............34
表二、GLOXD1啟動子區域甲基化程度之總表.................36
表三、ALDH1A2啟動子區域甲基化程度之總表................37
表四、CALCA啟動子區域甲基化程度之總表..................40
表五、DNMT3A1啟動子區域甲基化程度之總表................42
表六、DNMT3A2啟動子區域甲基化程度之總表................45
表七、候選基因p-value之總表………………………………………47

圖一、GLOXD1 PCR產物之膠體電泳圖.....................48
圖二、Pyrosequencing的原始結果圖....................49
圖三、細胞株裡GLOXD1啟動子區域甲基化程度...............50
圖四、組織檢體GLOXD1 PCR產物之膠體電泳圖...............51
圖五、組織檢體裡GLOXD1啟動子區域甲基化程度..............52
圖六、細胞株裡ALDH1A2 1st PCR 及Nested-PCR產物之電泳圖.53
圖七、細胞株裡ALDH1A2啟動子區域甲基化程度.................54
圖八、組織混合檢體裡ALDH1A2 1st PCR 及Nested-PCR產物之電泳圖..55
圖九、組織混合檢體裡ALDH1A2啟動子區域甲基化程度............56
圖十、個別組織檢體裡ALDH1A2 1st PCR 及Nested-PCR產物之電泳圖..57
圖十一、個別組織檢體裡ALDH1A2啟動子區域甲基化程度.........58
圖十二、細胞株裡CALCA PCR產物之膠體電泳圖...............59
圖十三、細胞株裡CALCA啟動子區域甲基化程度................60
圖十四、組織混合檢體裡CALCA PCR產物之膠體電泳圖...........61
圖十五、組織混合檢體裡CALCA啟動子區域甲基化程度...........62
圖十六、細胞株裡DNMT3A1 PCR產物之膠體電泳圖.............63
圖十七、細胞株裡DNMT3A1啟動子區域甲基化程度..............64
圖十八、組織混合檢體裡DNMT3A1 PCR產物之膠體電泳圖.........65
圖十九、組織混合檢體裡DNMT3A1啟動子區域甲基化程度..........66
圖二十、個別組織檢體裡DNMT3A1 PCR產物之膠體電泳圖..........67
圖二十一、個別組織檢體裡DNMT3A1啟動子區域甲基化程度........68
圖二十二、細胞株裡DNMT3A2 PCR產物之膠體電泳圖............69
圖二十三、細胞株裡DNMT3A2啟動子區域甲基化程度..............70
圖二十四、組織混合檢體裡DNMT3A2 PCR產物之膠體電泳圖........71
圖二十五、組織混合檢體裡DNMT3A2啟動子區域甲基化程度.........72

附錄一、亞硫酸鹽轉換（Bisulfite conversion）原理圖........73
附錄二、（A）甲基化專一性聚合酶鏈鎖反應（MS-PCR）與（B）亞硫酸鹽定序（Bisulfite sequencing）原理圖...74
附錄三、焦磷酸定序（Pyrosequencing）原理圖和冷光儀轉讀出的結果...75
附錄四、Pyrosequencing所需之Primer .........................76
附錄五、以候選基因ALDH1A2為例，做為Nested-PCR Primer之示意圖...77
附錄六、PyroMark Q24 Vacuum Workstation...................78
附錄七、Pyrosequencing分析GLOXD1與候選基因（ALDH1A2、CALCA、DNMT3A1與DNMT3A2）啟動子區域示意圖....79

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