利用酸鹼度敏感型雙子型界面活性劑製作之基因載體對核內體脂質膜結構之影響

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姓名

張時敏(Shih-Min Chang) 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

利用酸鹼度敏感型雙子型界面活性劑製作之基因載體對核內體脂質膜結構之影響
(Structural Disturbance on Endosomal Membranes by pH-Sensitive Gemini Surfactants as DNA Carriers)

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摘要(中)

許多疾病是由基因缺陷所引起，目前治療這類疾病最有潛力的方法之一，是利用非病毒載體壓實、包覆可修復基因缺陷之DNA，再將其投遞至細胞內以修復基因缺陷；雙子型界面活性劑便為其中一類極具發展性的非病毒載體。一般而言，載體與DNA所形成的複合物是由胞吞作用（endocytosis）進入細胞，在胞吞作用中所形成的核內體（endosome）會將複合物包覆在內，核內體的內部環境隨著發展過程逐漸轉變為酸性，以分解被胞吞之物質。因此，成功的載體必須在酸性環境下不被瓦解並同時保護DNA不被酵素分解，並由核內體逃脫（endosomal escape）以釋放出DNA達到轉染（transfection）的目標。過去的研究指出，在作為基因藥物載體時，酸鹼度敏感型雙子型界面活性劑較一般型雙子型界面活性劑擁有更佳的轉染效率。雖然科學家普遍推測此與前者擁有較佳的核內體逃脫能力有關，但卻無任何直接或間接之實驗證據證實此一推測，更不用說對其背後之分子機制的了解。因此，本研究利用UV光譜、原子力顯微鏡以及小角度X光散射，探討此兩類界面活性劑對改變DNA構型的能力，以及利用螢光光譜儀、小角度X光與中子散射，探討DNA由載體之釋放程度及複合物對仿核內體脂質囊泡在結構上的影響。藉由比較此兩類界面活性劑在各項性質上的差異，我們探究出酸鹼度敏感型雙子型界面活性劑擁有較高轉染效率的分子機制。

摘要(英)

Genetic diseases arise from abnormalities in genome. A promising approach in treating these diseases is employ a non-viral vehicle to compact and encapsulate therapeutic DNAs and deliver them into cells to repair the genetic disorders; gemini surfactants are among the most anticipated candidates for the gene delivery vehicle. Generally, the complexes formed by DNAs and vehicles enter cells by endocytosis. The endosomes formed during endocytosis encapsulate the complexes and become increasingly acidic to break down the internalized substances. As a result, a successful vehicle needs to maintain its structural integrity in an acidic environment to protect the DNAs from degradation and simultaneously escape from the endosome to release the DNAs for the transfection to occur. Earlier studies found that pH-sensitive gemini surfactants, when deployed as gene delivery vehicles, achieved higher transfection efficacies than their pH-insensitive counterparts. It is speculated that the higher transfection efficacy results from the former’s better capability to escape from endosomes. However, experimental evidence, whatever direct or indirect, remains lacking, let alone the understanding of the underlying molecular mechanisms. To address the issues, this study employs UV spectroscopy, atomic force microscope, and small-angle x-ray scattering to examine how the two types of gemini surfactants modify the conformations of DNAs; and employs fluorescence spectroscopy and small-angle x-ray and neutron scattering techniques to examine the extents of the DNA release from the vehicles and structural perturbations on the endosome-mimicking liposomes. Through comparing the differences in these aspects between the two types of geminni surfactants, we uncover the molecular mechanism accounting for the high transfection efficacy of the pH-sensitive gemini surfactants.

關鍵字(中)

★ 基因載體
★ 雙子型界面活性劑
★ 酸鹼度敏感型雙子型界面活性劑
★ 核內體逃脫

關鍵字(英)

論文目次

目錄...................................................................................................................X
圖目錄...........................................................................................................XIII
表目錄.........................................................................................................XVII
第一章緒論....................................................................................................1
1-1 基因治療............................................................................................1
1-1-1基因(Gene)........................................................................................1
1-1-2 基因治療....................................................................................4
1-2 常見非病毒載體的種類....................................................................8
1-2-1 陽離子脂質（cationic lipid）...................................................8
1-2-2 陽離子雙子型介面活性劑（cationic gemini surfactant）....12
1-2-3 酸鹼度敏感型陽離子雙子型介面活性劑（pH-sensitive cationic gemini surfactant）............................................................................14
1-3 胞外物質進入細胞的機制..............................................................18
1-3-1 進入細胞的方式......................................................................20
1-3-2 核內體逃脫（endosomal escape）.........................................22
1-4 研究目的與動機..............................................................................26
第二章實驗..................................................................................................27
2-1 實驗主要材料..................................................................................27
2-2 實驗相關藥品..................................................................................29
2-3 實驗儀器..........................................................................................31
2-4 樣品製備..........................................................................................32
2-4-1 陽離子雙子型界面活性劑製備..............................................32
2-4-2 緩衝溶液製備..........................................................................37
2-4-3 陽離子雙子型界面活性劑與DNA複合物製備.....................39
2-4-4 陰離子界面活性劑（SDS）解壓實複合物製備 ..................39
2-4-5 大型微脂體製備（Large Unilamellar Vesicles, LUV）........40
2-4-6 複合物與LUV混合樣品製備.................................................42
2-4-7 原子力顯微鏡樣品製備..........................................................43
2-5 分析儀器介紹與實驗測量方式......................................................44
2-5-1 核磁共振光譜（Nuclear Magnetic Resonance, NMR）.......44
2-5-2 質譜儀（Mass spectrometry）...............................................46
2-5-3 原子力顯微鏡（Atomic force microscopy, AFM）..............47
2-5-4 可見光紫外光分光光譜儀（UV-vis spectrophotometer）. 50
2-5-5 螢光分光光譜儀（Spectrofluorometer）..............................51
2-5-6 動態光散射（Dynamic light scattering, DLS）....................54
2-5-7 小角度X光散射（Small-Angle X-ray Scattering, SAXS） 55
2-5-8 小角度中子散射（Small-Angle Neutron Scattering, SANS） ..........................................................................................................................58
第三章結果與討論......................................................................................63
3-1 合成結果..........................................................................................63
3-1-1 陽離子雙子型界面活性劑（C12612）.................................63
3-1-2 酸鹼度敏感型雙子型界面活性劑（pHC12）......................66
3-1-3 界面活性劑性質......................................................................69
3-2 陽離子雙子型界面活性劑與DNA的作用力.................................73
3-2-1 利用UV吸收光譜探討陽離子雙子型界面活性劑在不同pH值環境下壓實DNA的能力.............................................................................73
3-2-2 利用原子力顯微鏡探討DNA壓實前後的結構變化.............79
3-2-3 利用小角度X光散射探討複合物形成前後的結構變化.......85
3-3 複合物添加陰離子界面活性劑後解壓實的情況..........................89
3-3-1 利用UV吸收光譜探討陽離子雙子型界面活性劑在不同pH值環境下解壓實DNA的能力.........................................................................89
3-4 仿核內體膜脂質囊泡與複合物間的作用力..................................96
3-4-1 利用螢光光譜儀探討在不同酸鹼值環境下複合物與LUV作用後DNA釋放的程度.....................................................................................97
3-4-2 利用小角度X光散射探討複合物與仿核內體膜的結構變化 ........................................................................................................................105
3-4-3 利用小角度中子散射探討仿核內體膜脂質囊泡與複合物混和前後的結構變化........................................................................................109
第四章結論................................................................................................114
參考文獻........................................................................................................118

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指導教授

陳儀帆

審核日期

2018-11-19

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