臺灣博碩士論文加值系統

L1TD1基因具有重複的”DNA轉座子長散在核元件”(Long interspersed nuclear element, LINE-1)核糖核酸結合區域，因此我們假設L1TD1會調控LINE-1啟動子的甲基化和整體甲基化的情形。在胃癌檢體及大腸癌的檢體中檢測到L1TD1不正常甲基化的情形，這代表了L1TD1在病理上有其重要性。為了瞭解L1TD1對於LINE-1的默化及體細胞基因體穩定性的維持之重要性，我們藉由CRISPR/Cas9系統將大腸癌、胃癌、乳癌以及人類間質幹細胞mesenchymal stem cells (MSCs)中L1TD1基因座進行剔除。我們發現細胞的表現型有顯著的改變且第三組織蛋白第九號位置離胺酸上的三甲基化修飾(tri-methylation of histone 3 at lysine 9, H3K9me3)的三甲基化被抑制進而影響LINE-1啟動子的甲基化。也藉由檢測L1TD1缺陷細胞的整體甲基化程度及HP1α的聚集情形，得知基因體的不穩定性增加及使間質幹細胞細胞的分化停滯。這個L1TD1所調控的表觀基因體修飾過程將如接下來所述: (1)L1TD1影響LINE-1甲基化程度; (2) L1TD1對於不同的細胞株中有其不同的甲基化程度的影響; (3) L1TD1會參與piRNA的生合成去影響細胞甲基化程度; (4) L1TD1影響細胞的分化。最後，L1TD1在沉默反轉錄轉座子及維持基因體的穩定性扮演重要的角色。

L1TD1 (LINE-1 Type Transposase Domain Containing 1) possesses two LINE-1 (long interspersed nuclear elements 1) RNA-binding domains and was hypothesized to regulate the methylation of LINE-1 promoter and the resulted methylome. Abnormal L1TD1 methylation was detected in gastric, colon and breast cancers and revealed the pathological importance of L1TD1 methylation. In order to verify the importance of L1TD1 in the silencing of LINE-1 and the maintenance of somatic genome stability, CRISPR/Cas9 was used to knockout (KO) the L1TD1 locus in colon, gastric, breast and mesenchymal stem cells (MSCs).We observed the changed cellular phenotype and the tri-methylation of histone 3 at lysine 9 was suppressed so as the methylation within LINE-1 promoter. The knockout also caused the loss of global methylation and the recruitment of HP1α, the genome stability was decreased and the differentiation of MSC was blocked. A L1TD1-mediated epigenomic modification pathway was then deduced. We figured: (1) L1TD1 regulates the methylation level of LINE-1; (2) L1TD1 is associated with the establishment of cell type-specific methylome; (3) L1TD1 takes part in the biogenesis of piRNA and impacts the methylome and the growth of cells; (4) L1TD1 deficiency interferes cell differentiation. At the end, L1TD1 silences retrotransposon and might help to maintain genome stability.

致謝 I
中文摘要 II
Abstract III
目錄 V
圖表目錄 IX
第一章 緒論 1
第一節 對位性基因體學 (Epigenetics) 1
第二節 去氧核醣核酸甲基化(DNA Methylation)與癌症的發生 2
第三節 LINE-1 對於基因穩定度的影響 4
第四節 LINE-1 甲基化對於基因穩定度的影響 8
第五節 L1TD1 對於基因穩定度的影響 9
第六節 實驗動機及目的 11
第二章 材料與方法 13
第一節 細胞株培養 13
第二節 CRISPR/Cas9基因剔除技術 14
第三節 細胞去氧核醣核酸萃取 (Cell Genomic DNA Purification) 14
第四節 去氧核醣核酸序列定序 15
第五節 核醣核酸萃取 (Total RNA Extraction) 17
第六節 核醣核酸濃度定量 (RNA Quantification) 18
第七節 合成單股互補去氧核醣核酸 (First-Strand cDNA Synthesis) 18
第八節 非編碼核糖核酸萃取 (Non-coding small RNA Extraction) 19
第九節 反轉錄聚合酶連鎖反應 20
第十節 即時半定量聚合酶鏈鎖反應 (semi-quantitative Real-Time Polymerase Chain Reaction) 21
第十一節 亞硫酸氫鹽轉化法 (Bisulfite Conversion) 23
第十二節 焦磷酸定序法 （Pyrosequencing） 24
第十三節 染色質免疫沉澱-定序Chromatin Immunoprecipitation sequencing (CHIP-Seq) 25
第十四節 Methyl-CpG-binding domain protein capture sequencing (MBD-Seq) 30
第十五節 西方墨點法 33
第十六節 免疫螢光染色 (Immunofluorescence Staining) 39
第十七節 誘導間質幹細胞分化成神經細胞 40
第十八節 誘導間質幹細胞分化成肝臟細胞 41
第十九節 Periodic Acid-Schiff (PAS)stain 醣類染色 42
第二十節 原子力顯微鏡分析（Atomic Force Microscope, AFM） 43
第三章 實驗結果 45
第一節 L1TD1基因座之剔除以及其驗證 45
第二節 L1TD1缺陷對於特定基因之piRNA生合成之影響 46
第三節 L1TD1對於細胞核內甲基化建立之影響 46
第四節 L1TD1對於LINE-1甲基化之影響 47
第五節 L1TD1對於整體基因體之甲基化影響 47
第六節 L1TD1Def細胞之CCCTC-binding factor (CTCF)於特定基因分布情形 48
第七節 L1TD1對於細胞染色質之影響 48
第八節 L1TD1對於基因體穩定性之影響 49
第九節 L1TD1對於MSC細胞分化情形之影響 49
第十節 L1TD1缺陷對於細胞型態之影響 51
第四章 討論 53
參考文獻 55
圖 一、各種L1TD1Def細胞株之驗證 60
圖 二、MSC、 HT29、AGS之L1TD1Def細胞株中piRNA的表現情形 61
圖 三、Suv39h1、H3K9me3在間質幹細胞 (Mesenchymal stem cells, MSCs)之免疫螢光染色分析 62
圖 四、焦磷酸定序偵測在乳癌細胞株 (MDA-MB-231)及胃癌細胞株 (AGS)中LINE-1啟動子上的甲基化程度 63
圖 五、間質幹細胞methyl-CpG binding domain-capture (MBD)定序實驗結果之Circos圖 64
圖 六、間質幹細胞染色體免疫共沉澱實驗結果之Circos圖 65
圖 七、L1TD1Def細胞之物理圖譜 (physical map) 68
圖 八、HP1α、Suv39h1在間質幹細胞之螢光免疫染色分析 70
圖 九、H2A.v、Lamin B在間質幹細胞之螢光免疫染色分析 71
圖 十、誘導間質幹細胞分化成神經細胞之螢光染色分析 72
圖 十一、誘導間質幹細胞分化成肝臟細胞之PAS染色分析及L1TD1表現情形 74
圖 十二、在L1TD1Def的MSC細胞株產生不正常的細胞型態 76
圖 十三、F-actin、α-tubulin、RassF1A在間質幹細胞之免疫螢光染色分析 78

表 一、本篇論文所使用驗證之引子序列 79
表 二、本篇論文所使用之引子序列 80
表 三、本篇論文所使用偵測肝臟分化細胞之引子序列 81

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