臺灣博碩士論文加值系統

端粒（telomere）是位於染色體末端由重複核酸序列組合而成的 結構，用於維持染色體的完整性，而端粒的長度會隨著細胞分裂或是 環境壓力而逐漸縮短，研究指出端粒長度的變化是導致人體衰老及癌 症產生的重要指針，因此端粒長度變化的測量是癌症預防的重要手段。 近年來報導了許多方法來測量端粒長度，例如末端限制性片段分析 （terminal restriction fragmentation，TRF），定量即時聚合酶連鎖反應 （quantitative real-time polymerase chain reaction，Q-PCR），定量螢光 原位雜合（quantitative fluorescence in situ hybridization，Q-FISH）及 流式細胞螢光原位雜合（flow cytometry and flow fluorescence in situ hybridization，Flow-FISH）等。這些方法都有各自的優點，但卻有一 個共通的弊病──無法測量線性化端粒DNA 的絕對長度，因此本實 驗發展利用超解析影像技術來觀測端粒的絕對長度。目前我們已經成 功將端粒DNA 線性沉積在疏水性玻片表面，透過端粒螢光標記探針 定位測量端粒絕對長度，同時我們也成功分離出單顆細胞DNA 並嘗 試將其完整的線性沉積在玻片上，希望未來能夠在單顆細胞的水平上 測量出細胞端粒長度。

Telomere is the structure composed by repeating nucleic acid sequences at the end of a chromosome, and which to maintain the integrity of the chromosome. The length of the telomere is gradually shortened when the cell divides or the environmental pressure. The study shows that the change of the telomere length is an important indicator of human aging and cancer. Therefore, the measurement of telomere length changes is crucial means of cancer prevention. In recent years, a lot of methods have been reported to measure the length of telomere, such as terminal restriction fragmentation (TRF), quantitative real-time polymerase chain reaction (QPCR), quantitative fluorescein Quantitative fluorescence in situ hybridization (Q-FISH) and flow cytometry and flow fluorescence in situ hybridization (Flow-FISH). All above the mentions, these methods have their own advantages, however, there is a common drawback—the absolute length of linearized telomere DNA cannot be measured. Hence, this experiment develops the techniques of super-resolution image to measure the absolute length of telomeres. We have successfully deposited telomere DNA on the surface of hydrophobic slides and measured the absolute length of telomeres through telomere fluorescent labeled probes at the present time. Simultaneously, we also achieved to isolate the DNA of a single cell and tried to completely deposit it on slides. It is hope that we can measure the telomere lengths at the level of single cell.

目錄
中文摘要 ...................................................................................................................................................I
Abstract ................................................................................................................................................... II
圖目錄 ..................................................................................................................................................... V
第一章 單分子螢光顯微鏡之簡介 ........................................................................................................ 1
1.1 緒論 ............................................................................................................................................... 1
1.2 螢光物理原理 ............................................................................................................................... 3
1.3 雷射掃描共軛焦顯微鏡 ............................................................................................................... 6
1.4 全內反射式螢光顯微鏡 ............................................................................................................... 8
1.5 超解析螢光顯微鏡 ..................................................................................................................... 12
1.5.1 受激放射耗乏顯微鏡技術 .................................................................................................. 12
1.5.2 單分子定位顯微鏡 .............................................................................................................. 18
1.5.2.1 光啟動定位顯微鏡 ...................................................................................................... 20
1.5.2.2 隨機光學重建顯微鏡 .................................................................................................. 23
1.5.2.3 直接隨機光學重建顯微術 .......................................................................................... 27
第二章 端粒之簡介 .............................................................................................................................. 33
2.1 端粒的發現 ................................................................................................................................. 33
2.2 人類端粒結構與功能 ................................................................................................................. 34
2.3 人類端粒結合蛋白 ..................................................................................................................... 37
2.3.1 TRF1 和TRF2 之功能 ........................................................................................................ 38
2.3.2 TIN2 之功能 ........................................................................................................................ 39
2.3.3 TPP1 和POT1 之功能 ........................................................................................................ 40
2.3.4 Rap1 之功能 ........................................................................................................................ 41
2.4 端粒長度維持機制 ..................................................................................................................... 42
2.4.1 端粒酶介導延長機制 ......................................................................................................... 42
2.4.2 端粒替代延長機制 ............................................................................................................. 43
2.5 端粒長度與衰老、癌症之關係.................................................................................................. 43
2.6 端粒長度檢測方法 ..................................................................................................................... 44
2.6.1 末端限制性片段分析 .......................................................................................................... 44
2.6.2 聚合酶連鎖反應法 .............................................................................................................. 46
2.6.3 定量螢光原位雜合 .............................................................................................................. 47
2.6.4 流式細胞螢光原位雜合 ...................................................................................................... 48
第三章 以超解析影像技術觀測端粒長度 .......................................................................................... 50
3.1 研究動機 ..................................................................................................................................... 50
3.2 實驗材料與方法 ......................................................................................................................... 50
3.2.1 實驗試藥 ............................................................................................................................. 50
3.2.2 螢光顯微鏡系統架構 .......................................................................................................... 52
3.2.3 實驗方法 ............................................................................................................................. 57
3.2.3.1 玻片製備 ...................................................................................................................... 57
3.2.3.2 細胞培養 ...................................................................................................................... 58
3.2.3.3 中期細胞染色體製備 .................................................................................................. 58
3.2.3.4 Genomic DNA 萃取 ..................................................................................................... 59
3.2.3.5 液珠蒸發線性沉積DNA ........................................................................................... 60
3.2.3.6 液珠平移線性沉積DNA ........................................................................................... 60
3.2.3.7 以螢光原位雜合法定位染色體端粒 ......................................................................... 60
3.2.3.8 以螢光原位雜合法定位端粒DNA ............................................................................ 62
3.2.3.9 以微米毛細管提取單顆細胞DNA ............................................................................ 64
3.2.3.10 破壞細胞之細胞膜與細胞核膜 ................................................................................ 65
3.2.3.11 以微米毛細管分離單顆細胞 .................................................................................... 65
3.3 結果與討論 ................................................................................................................................. 66
3.3.1 以螢光原位雜合法定位染色體端粒 .................................................................................. 66
3.3.2 比較不同表面修飾玻片影響DNA 線性沉積 ................................................................... 69
3.3.3 比較不同沉積方法影響DNA 線性沉積 ........................................................................... 70
3.3.4 以液珠平移沉積不同濃度λDNA ...................................................................................... 75
3.3.5 以螢光原位雜合法定位端粒DNA .................................................................................... 78
3.3.6 以微米毛細管提取單顆細胞DNA .................................................................................... 84
3.3.7 破壞細胞之細胞膜與細胞核膜 .......................................................................................... 84
3.3.8 以微米毛細管分離單顆細胞 .............................................................................................. 86
3.3.9 單顆細胞DNA 萃取 ........................................................................................................... 89
3.3.10 單顆細胞DNA 片段化 ..................................................................................................... 91
3.3.10.1 高溫剪切法 ................................................................................................................ 92
3.3.10.2 超音波剪切法 ............................................................................................................ 92
3.3.10.3 金屬離子催化剪切法 ................................................................................................ 96
3.3.10.4 限制性內切酶消化法 .............................................................................................. 104
3.4 結論與未來展望 ....................................................................................................................... 107
3.5 參考文獻 ................................................................................................................................... 108

圖目錄
圖1-1 Jablonski 模型 ................................................................................. 5
圖1-2 共軛焦顯微鏡裝置示意圖 ............................................................ 7
圖1-3 螢光顯微鏡照射系統示意圖 ........................................................ 9
圖1-4 光在不同介質之折射與全反射示意圖 ...................................... 10
圖1-5 物質與電磁波相互作用之能階變化示意圖 .............................. 13
圖1-6 受激放射耗乏顯微鏡運作原理示意圖 ...................................... 17
圖1-7 光啟動定位顯微鏡運作原理示意圖 .......................................... 22
圖1-8 Cy5 螢光分子之光轉化示意圖 ................................................ 25
圖1-9 隨機光學重建顯微運作原理示意圖 .......................................... 26
圖1-10 dSTORM 螢光分子光轉換機制 ................................................ 30
圖2-1 端粒結構示意圖 .......................................................................... 35
圖3-1 螢光顯微鏡系統裝置圖 .............................................................. 53
圖3-2 100X 物鏡下之微米尺 ................................................................ 55
圖3-3 100X 物鏡下之λDNA ................................................................. 56
圖3-4 以螢光原位雜合法定位染色體端粒實驗流程圖 ...................... 61
圖3-5 以螢光原位雜合法定位端粒DNA 實驗流程圖 ....................... 63
圖3-6 以螢光原位雜合法定位染色體端粒 .......................................... 67
圖3-7 比較不同表面修飾玻片影響DNA 線性沉積 ........................... 71
圖3-8 以液珠蒸發沉積法拉伸DNA .................................................... 73
圖3-9 於不同表面修飾玻片上觀察液珠接觸角 .................................. 74
圖3-10 以液珠平移法沉積λDNA ........................................................ 76
圖3-11 以液珠平移法沉積不同濃度λDNA ........................................ 77
圖3-12 比較DNA 樣品於EDC 固定前後與Blocking 前後之差異 .. 79
圖3-13 以傳統螢光顯微鏡系統觀測不同長度之端粒DNA .............. 82
圖3-14 比較傳統顯微鏡端粒DNA 影像與dSTORM 超解析端粒DNA
影像之差異 ............................................................................................... 83
圖3-15 嘗試以微米毛細管吸取單顆細胞DNA .................................. 85
圖3-16 破壞細胞之細胞膜與細胞核膜 ................................................ 87
圖3-17 以內徑為50μm 之微米毛細管分離單顆細胞 ......................... 88
圖3-18 單顆細胞DNA 萃取 ................................................................. 90
圖3-19 95℃加熱30 分鐘片段化DNA ................................................. 93
圖3-20 95℃加熱1 小時片段化DNA ................................................... 94
圖3-21 95℃加熱2 小時片段化DNA ................................................... 95
VII
圖3-22 超音波剪切法片段化DNA ...................................................... 97
圖3-23 以1 mM 銅離子催化切割DNA ............................................... 99
圖3-24 以5 mM 銅離子催化切割DNA ............................................. 100
圖3-25 三聯吡啶氯化釕六水合物之吸收圖 ...................................... 101
圖3-26 以1 mM 三聯吡啶釕(II)催化切割DNA ............................... 102
圖3-27 以5 mM 三聯吡啶釕(II)催化切割DNA ............................... 103
圖3-28 以限制性內切酶消化單顆細胞DNA 之TIRF 螢光顯微鏡影像
圖 ............................................................................................................. 106

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