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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周文堅 | |
| dc.contributor.author | Yueh-Chwen Hsu | en |
| dc.contributor.author | 徐悅淳 | zh_TW |
| dc.date.accessioned | 2021-06-08T02:43:14Z | - |
| dc.date.copyright | 2018-02-22 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-01-29 | |
| dc.identifier.citation | 1. Fisher, C.L., et al., Additional sex combs-like 1 belongs to the enhancer of trithorax and polycomb group and genetically interacts with Cbx2 in mice. Dev Biol, 2010. 337(1): p. 9-15.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20249 | - |
| dc.description.abstract | Additional sex combs-like 1（ASXL1）基因突變常發生在骨髓性惡性血液腫瘤患者。近期小鼠模式研究發現，針對血液系統剔除Asxl1或過量表現突變型ASXL1皆可在小鼠身上造成類似骨髓化生不良症候群之疾病。然而符合「生理劑量」之突變型ASXL1對實際生理之影響目前仍不清楚。為了探討此一問題，我們依最常在病人身上見到的ASXL1突變型式產製一帶有Asxl1突變的基因剔入鼠，並探討其對小鼠造血系統的病理生理作用機轉之影響。
經由鵝卵石區域形成細胞分析及聚落形成細胞分析我們發現帶突變型Asxl1之異型合子骨髓細胞具有較高的體外增殖能力。另一方面，在骨髓移植試驗中帶突變型Asxl1之異型合子小鼠的捐贈細胞在骨髓中所佔之比例在受贈者小鼠體內下降得比較快，顯示其長期體內再生能力受到損害。終其一生，帶有Asxl1突變之小鼠沒有明顯傾向會發生任何形式的血液病，這說明單獨只有Asx1突變不足引發白血病或其它惡性血液疾病。然而在帶有此突變之骨髓細胞中過量表現MN1可增加異體骨髓中增殖的能力，這揭示了Asxl1突變在疾病進程中可能扮演輔助的角色。經由分析ASXL1突變型及野生型人類白血病細胞以及帶有或不帶有MN1過量表現之Asxl1突變型及野生型小鼠造血幹細胞及造血前驅細胞之基因表現圖譜，我們發現Asxl1突變與缺氧、多潛能前驅細胞、造血幹細胞、KRAS及MEK基因組有關聯，ChIP-Seq分析並揭示了骨髓前趨細胞中受Asxl1突變調控之3甲基化組蛋白3離胺酸27(H3K27me3)圖譜。 在本研究中我們提出了第一個Asxl1突變基因剔入鼠模式，儘管此突變不足以導致惡性血液疾病，但是我們的研究結果顯示突變Asxl1降低了帶MN1過量表現骨髓細胞之移植門檻，並且在正常生理及血液疾病狀況下展現出特有的生物功能。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-08T02:43:14Z (GMT). No. of bitstreams: 1 ntu-107-D99421005-1.pdf: 3634079 bytes, checksum: 73d260f8e29bb7035a7265febb6b8b28 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii Acknowledgements iii 目錄 iv 圖表目錄 vii 中英文及縮寫對照表 x 摘要 xiii Abstract xiv 第1章：緒論 1 1.1 Additional sex combs-like 1 (ASXL1)簡介 1 1.2 多梳抑制蛋白家族(Polycomb repressor complex，PRC)之功能 2 1.3 植物同源異型功能區塊(Plant homeodomain，PHD) 5 1.4 急性骨髓性白血病(Acute myeloid leukemia)簡介 5 1.5 ASXL1突變和急性骨髓性白血病之關係 7 1.6 ASXL1功能相關之研究 9 1.7 前人研究之爭議及侷限 12 1.8 本研究之目標及研究成果 13 第2章：材料與方法 15 2.1 產製帶Asxl1突變之基因剔入鼠 15 2.2 帶Asxl1突變基因剔入鼠之繁殖計劃 15 2.3 鵝卵石區域形成細胞分析(CAFC) 15 2.4 聚落形成細胞分析(CFC) 16 2.5 小鼠骨髓移植實驗及競爭性再生單位分析(Competitive repopulating unit assay，CRU) 16 2.6 流式細胞分析及細胞分選 17 2.7 細胞週期分析 17 2.8 體外BrdU攝入分析 18 2.9 利用反轉錄病毒(Retrovirus)將過量表現MN1載體轉導至小鼠骨髓細胞中 19 2.10 長期培養起始細胞分析(Long-term culture initiating cell assay，LTC-IC) 19 2.11 以序列稀釋分析具MN1過量表現骨髓細胞在活體內重組再生之能力 19 2.12 微陣列(Microarray)分析 20 2.13 以組蛋白萃取及西方墨點法分析小鼠骨髓細胞中H3K27me3之含量 20 2.14 ChIP-Seq數據分析 20 第3章：實驗結果 22 3.1 產製Asxl1 G643WfsX12基因剔入鼠 22 3.2 Asxl1 G643WfxX12在小鼠中作用之區域 22 3.4 Asxl1 G643WfsX12異型合子之骨髓細胞有較野生型強的體外增生能力 22 3.5 Asxl1 G643WfsX12異型合子骨髓細胞在體內再生能力較野生型差 23 3.6 Asxl1 G643WfsX12異型合子骨髓細胞自我更新能力較野生型差 24 3.7 Asxl1 G643WfsX12異型合子骨髓中造血幹細胞及造血前驅細胞組成和野生型無顯著差異 24 3.8 單獨Asxl1 G643WfsX12不足以在小鼠引起血液疾病 25 3.9 Asxl1 G643WfxX12同型合子對小鼠不致命唯存活率不符合遺傳定律 26 3.10 Asxl1 G643WfsX12加上喪失功能Runx1突變無法在小鼠身上引發血液疾病 27 3.11 Asxl1 G643WfsX12降低具MN1過量表現之骨髓細胞被移植進入受贈者體內之門檻 29 3.12 微陣列分析顯示Asxl1突變及MN1過量表現之協同作用情形 31 3.13 GSEA顯示了Asxl1突變與MN1過量表現間交互作用所致的致癌基因功能 32 3.14 經由分析ChIP-Seq資料可發現Asxl1突變會改變H3K27me3的鍵結模式 32 第4章：討論 35 4.1 Asxl1 G643WfsX12基因剔入小鼠模式之臨床意義 35 4.2 體內和體外實驗結果差異的可能原因 35 4.3 總結異型合子Asx11突變與MN1過量表現間的關係 36 4.4 結合異型合子Asxl1突變及第二擊之其它基因突變 37 4.5 獲得突變型Asxl1 G643WfsX12同型合子之困難處 38 4.6 編輯小鼠基因表現之方法及實務上之侷限 40 4.7 Asxl1 G643WfsX12基因剔入鼠之基因表現圖譜 44 4.8 突變型Asxl1異型合子改變了H3K27me3的DNA結合圖譜 46 第5章：展望 47 5.1 Asxl1 G643WfsX12基因剔入鼠在白血病致病機轉可進行之後續研究 47 5.2 Asxl1 G643WfsX12基因剔入鼠在藥物篩選之可能應用 47 5.3 突變型Asxl1同型合子比例偏差之分析 48 5.4 造成突變型Asxl1仔鼠死亡原因之可能分析計劃 48 5.5 Asxl1 G643WfsX12突變對小鼠正常生理或/及行為之影響 50 Summary 52 參 考 文 獻 61 表1. 在Asxl1 G643WfsX12異型合子互配之繁殖計劃下同型合子仔鼠離乳數目及各基因型仔數所佔之百分比 70 表2. 在皆有MN1過量表現之背景下，移植異型合子 Asxl1突變型或野生型骨髓細胞之受贈者總數及移植成功之受贈者數目。 71 表3. H3K27me3峰值所在位置表現量有下降之基因列表。 72 圖1. 產製Asxl1 G643WfsX12基因剔入鼠。 74 圖2. 在異型合子互配之條件下胎兒的基因型分佈分析。 75 圖3. 鵝卵石區域形成細胞分析(CAFC)分析顯示帶異型合子突變型Asxl1之骨髓細胞形成鵝卵石區域的能力較野生型細胞更強。 76 圖4. 利用聚落形成細胞分析(CFC)分析異型合子Asxl1突變型骨髓細胞形成各形態成熟血球細胞細胞聚茖之能力。 77 圖5. 小鼠骨髓移植實驗及競爭性再生單位分析(competitive repopulation unit assays)。 78 圖6. 利用流式細胞儀分析受贈者骨髓中衍生自異型合子Asxl1突變型或野生型細胞之組成情形。 79 圖7. 以序列骨髓移植試驗分析異型合子Asxl1突變對造血幹細胞自我再生能力之影響。 80 圖8. 利用流式細胞儀分析異型合子Asxl1突變型及野生型小鼠骨髓中造血幹細胞及造血前驅細胞之組成情形。 81 圖9. 以流式細胞儀分析異型合子Asxl1突變型及野生型小鼠骨髓中造血幹細胞及造血前驅細胞之細胞週期。 82 圖10. 野生型及異型合子突變型Asxl1小鼠之體重記錄。 83 圖11. 年老(18個月)且帶異型合子Asxl1突變之公鼠其白血球(左圖)及紅血球(右圖)讀數會較同年齡之野生型公鼠來得高。 84 圖12. 利用流式細胞儀分析年老小鼠之骨髓組成。 85 圖13. 利用流式細胞儀分析年老異型合子Asxl1突變型及野生型小鼠骨髓、脾臟及週邊血之組成情形。 86 圖14. 利用流式細胞儀分析年老異型合子Asxl1突變型及野生型小鼠骨髓中造血幹細胞及造血前驅細胞所占之比率。 87 圖15. 年老Asxl1同型合子小鼠之全血計數讀值。 88 圖16. 年老Asxl1同型合子小鼠週邊血之全血計數讀值。 89 圖17. 年老Asxl1同型合子小鼠之體重、骨髓及脾臟之組成。 90 圖18. 利用流式細胞儀分析年老Asxl1同型合子小鼠骨髓中造血幹細胞及造血前驅細胞之組成形。 91 圖19. 產製喪失功能Runx1突變異型合子基因剔入鼠之示意圖。 92 圖20. 三個月齡Asxl1突變Runx1突變雙重異型合子剔入鼠之全血球計數讀。 93 圖21. 利用流式細胞儀分析Asxl1突變Runx1突變雙重異型合子剔入鼠之骨髓組成。 94 圖22. 以CFC分析Asxl1突變Runx1突變雙重異型合子剔入鼠之骨髓組成。 95 圖23. 序列骨髓移植試驗分析Asxl1突變Runx1突變雙重異型合子剔入鼠骨髓細胞之自我更新能力。 96 圖24. 十八個月齡Asxl1突變Runx1突變雙重異型合子剔入鼠全血球計數分析。 97 圖25. 年老Asxl1突變Runx1突變雙重剔入鼠之血液器官及體型分析。 98 圖26. 突變型ASXL1及MN1高表現之交互作用情形。 99 圖27. 體外分析帶MN1過量表現之突變型Asxl1骨髓細胞活性。 100 圖28. 序列稀釋骨髓移植實驗顯示在MN1過量表現之Asxl1突變骨髓細胞有較高比例之細胞具有在受贈者身上進行骨髓重建之能力。 101 圖29. ASXL1突變與MN1過量表現間之交互作用的基因表達圖譜及基因組分析。 102 圖30. 小鼠骨髓細胞及AML患者樣本間皆有富集之基因組GSEA圖。 103 圖31. 異型合子Asxl1突變無法在骨髓細胞中顯著改變H3K27me3的量。 104 圖32. 將異型合子突變型及野生型Asxl1專屬H3K27me3峰值利用IGV視覺化之結果。 105 圖33. 在扣除兩者皆有的讀取峰值後異型合子突變型及野生型Asxl1之專屬峰值在基因體中分佈之情形。 106 圖34. 利用MEME-ChIP web tool所分析出來異型合子突變型及野生型Asxl1專屬峰值所涵蓋區域最常見之DNA序列。 107 圖35. 微陣列分析中所發現表現量下降的基因和H3K27me3讀取峰值之關聯情形。 108 附錄： 109 一. 個人在碩博士班修業期間發表之相關論文： 109 二. 抗體列表： 110 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討ASXL1突變在惡性血液腫瘤所扮演的角色 | zh_TW |
| dc.title | Exploring the role of ASXL1 mutation in myeloid malignancies | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 陳佑宗 | |
| dc.contributor.oralexamcommittee | 楊偉勛,黃凱文,林亮音,林淑華,張原翊 | |
| dc.subject.keyword | Asxl1,MN1,造血幹細胞,異體骨髓增殖,生理劑量, | zh_TW |
| dc.subject.keyword | Asxl1,MN1,hematopoietic stem cell,engraftment,physiological dose, | en |
| dc.relation.page | 111 | |
| dc.identifier.doi | 10.6342/NTU201800229 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2018-01-30 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 臨床醫學研究所 | zh_TW |
| 顯示於系所單位： | 臨床醫學研究所 | |
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