臺灣博碩士論文加值系統

去氧核醣核酸甲基化是細胞中常見的一種對位性基因修飾，正常的去氧核醣核酸甲基化用來調控細胞命運，而不正常的去氧核醣核酸甲基化則會改變正常細胞。然而，甲基化低下會導致基因組不穩定並且加速細胞轉化；而抑制腫瘤基因如HIC1及RassF1A的去氧核醣核酸有甲基化增加現象亦足以將體幹細胞(Somatic stem cell)轉化為類癌幹細胞(Cancer stem cell-like cell)。因此，我們認為不正常的去氧核醣核酸甲基化導致基因的功能失去調控，可能是造成癌化的原因之一。先前的研究已知CD44及CD133為大腸直腸癌幹細胞的標記，我們實驗室利用基因體微陣列(Microarray)的方式偵測HCT116 subpopulation中的甲基化差異。找出Endosulfine alpha這個基因在體幹細胞中為雙基因位點(bivalent locus)，我們認為該基因對於細胞命運調控扮演重要腳色。ENSA過去被認為是磺醯尿素類受體(Sulfonylurea receptor)的內因性配位體(Eendogenous ligand)，負責調控ATP敏感之鉀離子通道(ATP-sensitive potassium channel)，對於胰臟β細胞分泌胰島素的調控扮演重要角色。此外，在表達CD90的人類肝癌細胞(HepG2 cells)中ENSA啟動子呈現高度甲基化，但是在不表達CD90的HepG2 cells中則是呈現低度甲基化的現象。因此，我們認為ENSA基因的默化可能是影響肝癌細胞起始的關鍵之一，但是對於肝癌細胞的擴散卻不是那麼的必要。然而表達CD90的肝癌細胞非常之少，難以收集。於是我們嘗試從HCT116大腸直腸癌細胞中分離出表達CD133的細胞。利用甲基化特異性聚合酶連鎖反應偵測此細胞中ENSA啟動子的甲基化圖譜，發現到在表達CD133的HCT116細胞中ENSA啟動子呈現高度甲基化，而不表達CD133的細胞中則是低度甲基化的現象。接著透過及時定量聚合酶連鎖反應偵測ENSA的表達量，發現在不表達CD133的HCT116細胞中其ENSA表達量高於含CD133表達的細胞。該結果顯示，在大腸直腸癌細胞中ENSA對於癌細胞的調控功能與肝癌細胞中相似。因此，我們認為該基因ENSA的甲基化可能為大腸直腸癌發生的早期生物標記。近一步研究其可能作為大腸直腸癌腫瘤抑制基因的機制。

DNA methylation is one of several epigenetic changes observed in cells. Normal DNA methylation regulates cell fate, whereas abnormal DNA methylation transforms normal cells. A genetic hypomorphic model indicated that global hypomethylation could lead to the loss of genome stability and accelerate cellular transformation. In contrast, increased DNA methylation within tumor suppressor genes such as HIC1 and RassF1A is sufficient to transform a somatic stem cell into cancer stem cell-like (CSC-like) cells. Taken together, we suggest that associated abnormal DNA methylation indicates dysregulated gene function, which might be associated with tumorigenesis. Previously, CD44 and CD133 in particular have been identified as markers for stem cells in colon cancer. Our laboratory used the microarray to detect the methylation differences between the HCT116 subpopulation. One of the genes, ENSA is a bivalent locus in somatic stem cells, suggesting its critical role in cell fate determination. Endosulfine alpha (ENSA) was initially identified as an endogenous ligand of sulfonylurea receptor associated with ATP-dependent potassium channels in pancreatic beta cells. Thus, ENSA is a regulator of insulin secretion. Furthermore, the ENSA promoter is hypermethylated in CD90-expressing HepG2 cells but is hypomethylated in CD90 non-expressing HepG2 cells. We speculate that silenced ENSA function might be essential for liver cancer initiating cells but is not necessary for tumor expansion in liver cancer. But CD90 cells are very rare, so we try to use the CD133 mark to isolated CSC-like cells from HCT116 cells. Through methylated PCR(MSP) analysis, I discovered that the ENSA promoter is hypermethylted in CD133-expressing HCT116 cells but is hypomethylated in CD133-nonexpressing HCT116 cells. Through reverse transcription PCR (RT-PCR) analysis, the CD133-nonexpressing HCT116 cells expressing level is higher than CD133-expressing HCT116 cells. We suggest that the ENSA methylation is an early biomarker in colon cancer initiating cells and confirm that ENSA is a tumor suppressor gene in colon cancer.

中文摘要...i
英文摘要...iii
目錄...v
圖表目錄...vii
第一章 緒論
第一節 大腸直腸癌...1
第二節 癌症幹細胞...3
第三節 癌症對位性基因體學...4
第四節 去氧核醣核酸甲基化...6
第二章 材料與方法
第一節 細胞培養...8
第二節 癌幹細胞分離...8
第三節 染色體去氧核醣核酸萃取...9
第四節 核醣核酸萃取...9
第五節 反轉錄聚合酶連鎖反應...10
壹、 去氧核醣核酸酶處理...10
貳、 合成單股去氧核醣核酸...10
第六節 及時定量聚合酶連鎖反應...11
第七節 亞硫酸氫鹽轉換法...12
第八節 甲基化特異性聚合酶連鎖反應...12
第九節 免疫螢光染色...13
第十節 差異性甲基化晶片雜合反應...13
壹、 利用限制酵素MseI將去氧核醣核酸片段化...14
貳、 去氧核醣核酸片段接上H12/24連接引子對...14
參、 甲基化敏感酵素BstUI、HpaII剪切...14
肆、 利用聚合酶連鎖反應擴增帶有連接引子對之去氧核醣核酸片段...15
伍、 標定...15
第十一節 抑制甲基化藥物處理...16
第三章 實驗結果
第一節 癌症特異性的不正常去氧核醣核酸甲基化...17
第二節 ENSA啟動子之對位性基因狀態...18
第三節 患者樣本中之ENSA不正常甲基化觀察...20
第四節 過度表達ENSA衰弱肝癌細胞生長...20
第五節 驗證HCT116之CD133+類癌幹細胞ENSA甲基化狀態...21
第四章 討論...24
參考文獻...27
圖表...31

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