臺灣博碩士論文加值系統

經由過去的研究指出，癌化的過程中常伴隨著對位性基因體的修飾，例如在不同的癌症中，HIC1與RASSF1A，二個調控細胞週期(cell cycle)與細胞計畫性凋亡機制(apoptosis)之抑癌基因，會經由去氧核醣核酸甲基化修飾使其失去作用。但是目前還沒有任何研究發現，是否對位性基因體修飾就足以使細胞癌化。在我們的實驗中使用了量身訂作甲基化技術，將骨髓間質幹細胞中HIC1與RASSF1A共同甲基化，另一方面也將調控生長發育的Salvador-Warts-Hippo (SWH)訊息傳遞路徑中十個基因共同甲基化，觀察是否透過去氧核醣核酸甲基化修飾作用就足以使正常骨髓間質幹細胞癌化，並正向的去證實癌症幹細胞理論。實驗結果顯示，在骨髓間質幹細胞內將HIC1與RASSF1A共同甲基化(me_H&R)或將SWH訊息傳遞路徑中十個基因共同甲基化(me_SWH)會導致細胞失去接觸抑制作用。除此之外，me_H&R與me_SWH細胞株亦具有許多轉型細胞的特性，包含了me_H&R與me_SWH細胞比起對照組骨髓間質幹細胞具有較高的增生速率，並可於軟性瓊脂培養基(soft agar)內形成許多細胞群落。而轉型的me_H&R與me_SWH細胞依然具有幹細胞之特性，因其表達CD133表面抗原並能夠分化為神經細胞。而me-H&R細胞株更可在免疫缺乏之裸鼠體內形成腫瘤。綜合以上結果得知，利用量身訂作甲基化技術將抑癌基因HIC1與RASSF1A在骨髓間質幹細胞內高度甲基化，會使得骨髓間質幹細胞轉型為具有癌化性質的細胞，並且此細胞依然具有幹細胞之特性。在本實驗中我們證實了，去氧核醣核酸甲基化修飾足以使正常成體幹細胞轉型為癌細胞。

To evaluate if the epigenomic modifications are sufficient to transform stem cells and test the tumor stem cell hypothesis, we methylated the HIC1 and RassF1A (H&R) loci together or ten loci from the Salvador-Warts-Hippo (SWH) pathway in the senescent human mesenchymal stem cells (MSCs) to observe if the MSCs would be transformed. The SWH pathway is initially identified in the Drosophila model that loss of function of genes within this pathway accelerates tumorigenesis. Both HIC1 and RassF1A are tumor suppressor genes and are frequently silenced by DNA methylation in many types of cancer, thus, the knock-down of HIC1 and RassF1A is aimed to recapitulate the generally believed oncogenic processes. We found that transfection of in vitro methylated SWH or HIC1&RASSF1A into human MSCs did not alter the stemness of human MSCs, as stem cell markers like CD133 and Oct4 remained and these transfected MSCs can be induced to differentiate. However, the treated cells also exhibit several characteristics of tumor cells, including higher proliferation rates, greater colony formation capability, greater metastatic ability in trans-well studies and tumoral formation ability in nude mice. Taken together, these data indicate that human MSCs can be transformed by in vitro methylated HIC1 and RassF1A. Because the derived cells possess both the stem and tumoral properties, we would like to suggest that DNA methylation is sufficient to transform a somatic stem cell.

致謝……………………………………………………………………Ⅰ
中文摘要………………………………………………………………Ⅱ
英文摘要………………………………………………………………Ⅲ
目錄………………………………………………………………....Ⅳ
圖表目錄………………………………………………………………Ⅵ
附圖目錄………………………………………………………………Ⅷ
第一章 緒論
第一節 癌症對位性基因體學…………………………………………1
第二節 去氧核醣核酸甲基化(DNA methylation)………………….3
第三節 抑癌基因Hypermethylation In Cancer 1 (HIC1)概述….5
第四節 抑癌基因RASSF1A概述……………………………………….6
第五節 Salvador-Warts-Hippo (SWH)訊息傳遞路徑………………6
第六節 實驗目的………………………………………………………7
第二章 材料與方法
第一節 細胞培養………………………………………………………8
第二節 於骨髓間質幹細胞中將其抑癌基因HIC1、RASSF1A甲基化.8
第三節 基因選殖………………………………………………………9
第四節 活體外去氧核醣核酸甲基化 (In vitro DNA methylation)……10
第五節 甲基化去氧核醣核酸片段轉染(transfection)………….10
第六節 亞硫酸鹽轉化法 (bisulfite conversion)………………11
第七節 即時定量甲基化特異性聚合酶連鎖反應分析…………….11
第八節 免疫螢光染色分析………………………………………….12
第九節 軟瓊脂群落形成實驗 (soft agar colony formation assay)…13
第十節 細胞生長速度之檢測……………………………………….14

第十一節 誘導神經分化…………………………………………….14
第十二節Differential Methylation Hybridization (DMH) array………15
第十三節 DNA甲基化抑制劑(5-aza-2’-deoxycitydine)處理….17
第十四節 免疫組織螢光染色 (Tissue Immunostaining)……….17
第十五節 免疫組織化學染色 (Immunohistochemistry)…………18
第十六節 流式細胞儀(flow cytometry)分析…………………….19
第三章 實驗結果
第一節 抑癌基因HIC1、RASSF1A量身定做甲基化…………………20
第二節 me_H&R細胞株中有特定基因甲基化改變………………….22
第三節me_H&R與me_SWH細胞株仍具有幹細胞性質…………………24
第四節 me_H& R與me_SWH細胞株具有癌細胞之性質………………25
第五節 由me_H&R細胞株生成之腫瘤中有間質細胞(mesenchymal cells)轉型為上皮細胞的現象……………………………………..29
第六節 經由Xenograft會增加帶有CD133表面抗原細胞的比例….30
第七節 me_H&R細胞株具有較高的抗藥性………………………….31
第四章 討論………………………………………………………….32
參考文獻……………………………………………………………..35
圖表……………………………………………………………………42
附圖……………………………………………………………………69

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