博碩士論文 103324045 詳細資訊




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姓名 尤冠霖(Guan-Lin You)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 探討具有不同間隔長度的陰、陽離子雙子型界面活性劑對於DNA壓實與解壓實之影響
(Compaction and Decompaction of DNA by Cationic and Anionic Gemini Surfactants of Varying Spacer Lengths)
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摘要(中) 在傳遞DNA至人體細胞核以進行基因治療的過程中,DNA必須被壓實以降低遭受酵素分解或環境影響的機會,而因壓實而縮小之DNA亦較為容易進入至細胞核中。反之,DNA的被解壓實則是基因治療中的另一個必要步驟,以使DNA得以恢復原先之構型,並進行其基因表現之功能。傳統的單鏈型陽離子或陰離子界面活性劑對DNA的壓實與解壓實效果及其作為非病毒型基因載體的可能性經常是許多研究探討的主題。基於此一研究趨勢,本研究發展出一個以雙子型界面活性劑為基礎的新穎DNA壓實/解壓實系統。雙子型界面活性劑是由一條分子鏈將兩個單鏈型界面活性劑結合為一的一類界面活性劑;此一特殊的分子結構,使其比單鏈型界面活性劑具有更好的表面活性,並可藉由對橋接分子鏈的改變調整其分子結構及性質。在本研究中,我們使用了紫外光分光光譜儀、圓二色光譜儀、原子力顯微鏡及小角度X光散射等技術,探討不同的橋接分子鏈長度如何影響雙子型界面活性劑對於DNA壓實與解壓實之效果及其作用的機制。實驗數據顯示,陽離子與陰離子雙子型界面活性劑各自具有使DNA壓實與解壓實之能力,且橋接分子鏈之長度為6個碳數的雙子型界面活性劑對於DNA的壓實與解壓實效果皆較長度為3個碳數之雙子型界面活性劑為佳。我們推測,DNA壓實效果上的差異應與陽離子雙子型界面活性劑分子的離子化程度及其與DNA的複合自組裝結構的不同有關;而在DNA解壓實上的差異,我們則推測與陰離子雙子型界面活性劑的疏水性以及溶液中陰離子雙子型界面活性劑的帶電荷量有關。本研究因此以分子特性及超分子自組裝結構的角度,解釋了雙子型界面活性劑於DNA壓實/解壓實上的作用機制。
摘要(英) For an efficient delivey into the nuclei of human cells, the therapeutic DNAs of a gene therapy need to be compacted to protect them from the enzymatic hydrolysis or external chemical/biochemical stresses; the reduced DNA sizes by compaction also facilitate their delivery to the nuclei. On the other hand, decompaction of the compacted DNAs, which restores the DNAs to their native conformations and re-activates the gene expression, is a prerequisite for an effective gene therapy. Conventional cationic and anionic surfactants are often studied for their respective capabilities to compact and de-compact DNAs, with their potential of being a safe non-virus gene delivery system evaluated. Following this research interst, the present study develops a novel DNA compaction/decompaction system based on gemini surfactants. Gemini surfactants are dimers of two conventional surfactant molecules connected with a spacer. Due to this unique molecular structure, gemini surfactants commonly display superior surface activities than their conventional counterparts, with their molecular structure and material properties tunable via modulating the spacer. Here, we synthesize cationic and anionic gemini surfactants of varying spacer lengths to investigate how and why the change in the molecular structures of the gemini surfactants affects their DNA compaction/decompaction efficacy by using UV-vis spectrophotometer, dynamic light scattering, atomic force microscopy, circular dichroism and small-angle x-ray scattering. The gemini surfactants are proven competent in the DNA compact/decompaction, and the gemini surfactants with the 6-carbon long spacer are observed to display higher DNA compaction/decompaction efficiency than the one with the 3-carbon long spacer. The discrepancy in the DNA compaction efficacy might arise from the differences in the ionization degrees of the cationic gemini surfactants and in the self-assembled structures of the DNA-surfactant complexes. For the DNA decompaction, the difference in hydrophobicity and ionization of the anionic gemini surfactants may potentially lead to the difference in efficacy. The present study therefore explains the mechanisms underlying the DNA compaction/decompaction by gemini surfactants in terms of molecular property and supramolecular structure.
關鍵字(中) ★ 去氧核醣核酸
★ 雙子型界面活性劑
★ 壓實
★ 解壓實
關鍵字(英) ★ DNA
★ gemini surfactant
★ compaction
★ de-compaction
論文目次 中文摘要 .................................................................................................................................... I
英文摘要 ................................................................................................................................. III
誌謝辭 ...................................................................................................................................... V
目錄 ......................................................................................................................................... VI
圖目錄 ..................................................................................................................................... IX
表目錄 ................................................................................................................................... XII
化學式與化學名稱對照表 ................................................................................................... XIII
第一章 緒論 .............................................................................................................................. 1
1-1 基因治療.................................................................................................................... 3
1-1-1去氧核糖核酸(deoxyribonucleic acid, DNA) .......................................... 3
1-1-2 DNA在自然界中壓實(compaction)之行為 ........................................... 6
1-1-3 基因治療(gene therapy) .......................................................................... 8
1-2 非病毒型之DNA壓實劑(compaction agent)................................................... 14
1-2-1 多價陽離子(multivalent cations)與多胺(polyamines) .................... 14
1-2-2 陽離子脂質與微脂體(cationic lipids and liposomes) .......................... 18
1-2-3 陽離子界面活性劑(cationic surfactant) ............................................... 22
1-2-4 陽離子雙子型界面活性劑(cationic gemini surfactant) ....................... 28
1-3 陰離子界面活性劑與DNA複合物間的作用 ....................................................... 32
1-4 研究目的與動機...................................................................................................... 34
第二章 實驗 ............................................................................................................................ 35
2-1 實驗材料.................................................................................................................. 35
2-2 實驗藥品.................................................................................................................. 36
2-2 實驗器材.................................................................................................................. 38
2-3 實驗儀器.................................................................................................................. 39
2-4 陽離子雙子型界面活性劑(cationic gemini surfactant)之製備 ........................ 40
2-5 陰離子雙子型界面活性劑(anionic gemini surfactant)之製備 ......................... 43
2-6 DNA與雙子型界面活性劑(gemini surfactant)複合物之製備 ......................... 47
2-6-1 Tris-EDTA 緩衝溶液製備 ......................................................................... 47
2-6-2 DNA與陽離子界面活性劑(cationic gemini surfactant)複合物之製備 ............................................................................................................................... 47
2-6-3 DNA由陰離子雙子型界面活性劑(anionic gemini surfactant)解壓實(decompaction)之製備 ..................................................................................... 48
2-7 分析儀器介紹.......................................................................................................... 50
2-7-1核磁共振光譜(Nuclear Magnetic Resonance Spectroscopy, NMR) ..... 50
2-7-2質譜儀(Mass spectrometry) ................................................................... 54
2-7-3可見光紫外光分光光譜儀(UV-vis spectrophotometer) ....................... 56
2-7-4圓二色光譜儀(Circular dichroism spectroscopy, CD) .......................... 58
2-7-5原子力顯微鏡(Atomic force microscopy, AFM) .................................. 62
2-7-6小角度X光散射(Small-Angle X-ray Scattering) ................................. 65
2-7-7動態光散射儀(Dynamic light scattering, DLS) ..................................... 69
第三章 結果與討論 ................................................................................................................ 70
3-1 DNA與陽離子雙子型界面活性劑間的作用 ......................................................... 70
3-1-1使用UV吸收光譜探討陽離子雙子型界面活性劑對DNA壓實之效果 70
3-1-2使用圓二色光譜儀探討DNA被陽離子雙子型界面活性劑壓實時二級結構之變化 ............................................................................................................... 74
3-1-3使用原子力顯微鏡探討DNA被陽離子雙子型界面活性劑壓實後之形貌 ............................................................................................................................... 76
3-1-4使用小角度X光散射探討DNA被陽離子雙子型界面活性劑壓實後之排列結構 ................................................................................................................... 78
3-1-5使用動態光散射儀探討DNA被陽離子雙子型界面活性劑壓實時複合物大小之變化 ........................................................................................................... 84
3-2 DNA與陰離子雙子型界面活性劑間的作用 ......................................................... 86
3-2-1使用UV吸收光譜探討陰離子雙子型界面活性劑對DNA複合物解壓實之效果 ................................................................................................................... 86
3-2-2使用圓二色光譜儀探討DNA複合物被陰離子雙子型界面活性劑解壓實時二級結構之變化 ............................................................................................... 88
3-2-3使用原子力顯微鏡探討DNA被陰離子雙子型界面活性劑解壓實後之形貌 ........................................................................................................................... 89
3-2-4使用動態光散射儀探討DNA複合物被陰離子雙子型界面活性劑解壓實時複合物大小之變化 ........................................................................................... 90
3-3不同陽離子雙子型界面活性劑之spacer長度與DNA間作用關係 .................... 91
3-3-1 不同spacer長度對於DNA壓實效果之影響 .......................................... 91
3-3-2 DNA的結構變化與沉澱可能之關係 ........................................................ 96
3-3-3 DNA與陽離子雙子型界面活性劑形成之自組裝結構與DNA壓實效果及DNA轉染效率間之關係探討 ......................................................................... 97
3-4不同陰離子雙子型界面活性劑之spacer碳數對於DNA複合物解壓實之影響 ...................................................................................................................................... 103
第四章 結論 .......................................................................................................................... 109
參考文獻 ............................................................................................................................... 111
附錄 ....................................................................................................................................... 121
1. 核磁共振光譜 .......................................................................................................... 122
1-1 1H NMR Spectrum of C16316 ...................................................................... 122
1-2 1H NMR Spectrum of C16616 ...................................................................... 122
1-3 1H NMR Spectrum of A848 .......................................................................... 123
1-4 13C NMR Spectrum of A848 ......................................................................... 123
1-5 1H NMR Spectrum of A868 .......................................................................... 124
1-6 13C NMR Spectrum of A868 ......................................................................... 124
2. 質譜儀數據 .............................................................................................................. 125
2-1 C16316 .......................................................................................................... 125
2-2 C16616 .......................................................................................................... 126
2-3 A848 .............................................................................................................. 127
2-4 A868 .............................................................................................................. 128
3. 紫外光光譜儀數據 .................................................................................................. 129
3-1 C16316對Salmon sperm DNA之壓實作用 ............................................... 129
3-2 C16616對Salmon sperm DNA之壓實作用 ............................................... 131
3-3 A848對DNA-C16316之解壓實作用 ......................................................... 133
3-4 A848對DNA-C16616之解壓實作用 ......................................................... 135
3-5 A868對DNA-C16316之解壓實作用 ......................................................... 137
3-6 A868對DNA-C16616之解壓實作用 ......................................................... 139
4. 圓二色光譜儀數據 .................................................................................................. 142
4-1 C16316對Salmon sperm DNA之壓實作用 ............................................... 142
4-2 C16616對Salmon sperm DNA之壓實作用 ............................................... 145
4-3 A848對DNA-C16316之解壓實作用 ......................................................... 148
4-4 A868對DNA-C16316之解壓實作用 ......................................................... 150
4-5 A848對DNA-C16616之解壓實作用 ......................................................... 152
4-6 A868對DNA-C16616之解壓實作用 ......................................................... 154
5. 小角度X光散射數據 ............................................................................................. 156
5-1 C16316與Lambda DNA之壓實作用 ......................................................... 156
5-2 C16616與Lambda DNA之壓實作用 ......................................................... 157
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指導教授 陳儀帆(Yi-Fan Chen) 審核日期 2016-8-3
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