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研究生:陳家豪
研究生(外文):Chia-Hao Chen
論文名稱:利用改質蒙脫石當作基因傳送之載體
論文名稱(外文):Modified Montmorillonite as Nonviral Vector for Gene Delivery
指導教授:林峰輝林峰輝引用關係
指導教授(外文):Feng-Huei Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:57
中文關鍵詞:蒙脫石十六烷基三甲基胺基因傳送系統
外文關鍵詞:montmorillonitehexadecyltrimethylammoniumgene delivery system
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目前用來傳遞基因的載體主要分為病毒性載體和非病毒性載體。病毒性載體雖然有相當好的基因傳遞效果,但是會引起體內的免疫反應,也可能因病毒本身引發的突變,而造成毒性;非病毒性載體雖然不具細胞毒性,製備上也較為簡單,但是其傳遞效果卻不盡理想。上述因素均是目前基因治療所遭遇的瓶頸,為了克服上述問題,本研究希望能夠利用改質過的無機黏土礦物當做基因載體,來評估其基因傳送之可行性。

本研究主要利用十六烷基三甲基胺(hexadecyltrimethylammonium, HDTMA)對具有層狀結構的蒙脫石(montmorillonite, MMT)進行改質,而撐開蒙脫石的層間(interlayer),並將線性(linear)和質體(plasmid)DNA與改質後的蒙脫石製備成兩種不同DNA結構的複合物。本研究中製備DNA/HDTMA-蒙脫石複合物的適當條件為:
A.反應時間:2小時
B.改質蒙脫石的重量比例:HDTMA-MMT/DNA=50
C.HDTMA-MMT改質程度:1.5CEC
D.反應之pH值:10.7

由實驗結果發現,研究中製備的DNA/HDTMA-蒙脫石複合物,可以成左犒幭硅�姆細胞進行轉染,使其表現綠色螢光。
At the present time, major gene delivery systems employ either vrial or non-viral vectors. In general, viruses can mediate efficient gene transfer. However, there may provoke mutagenesis and carcinogenesis. Considering these limitations, non-viral vectors have several advantages over viral vectors for gene therapy. However, non-viral vectors are still relatively inefficient.

The aim of this study is to develop an effective non-viral gene transfer system by the combination of cationic hexadecyltrimethylammonium(HDTMA) and montmorillonite(MMT). Montmorillonite is composed of silicate sheets with adsorbed exchangeable cations. The intercalation with organic materials increases the spacing between the silicate sheets. In the study, we try to find the optimal condition for the preparation two kinds of pEGFP-C1/HDTMA-MMT composites. The optimal condition for the preparation of composite in this study is 1mg of 1.5CEC HDTMA-MMT under a pH value of 10.7, and with a soaking time for 2h. Preparation of plasmid DNA / 1.5CEC-HDTMA-MMT in pH value=10.7 was proved with capacity of gene delivery.
目錄
摘要--------------------------------------------------------------------------------------Ⅰ
英文摘要--------------------------------------------------------------------------------Ⅱ
目錄-------------------------------------------------------------------------------------Ⅲ
圖目錄--------------------------------------------------------------------------------Ⅵ
表目錄--------------------------------------------------------------------------------Ⅷ
符號說明--------------------------------------------------------------------------------Ⅸ

第一章 緒論----------------------------------------------------------------------------1
1.1 前言------------------------------------------------------------------------------1
1.2 基因傳送系統------------------------------------------------------------------1
1.2.1 病毒性載體----------------------------------------------------------------1
(a) 反轉錄病毒載體--------------------------------------------------------2
(b) 腺病毒載體--------------------------------------------------------------2
(c) 單純皰疹病毒載體-----------------------------------------------------3
(d) 腺相關病毒載體--------------------------------------------------------4
(e) 人類反轉錄病毒載體--------------------------------------------------5
1.2.2 非病毒性載體-------------------------------------------------------------5
(a) 微脂粒--------------------------------------------------------------------6
(b) DNA直接傳送法-------------------------------------------------------6
(c) DNA-蛋白質複合物--------------------------------------------------7
(d) 帶正電之高分子奈米微粒-------------------------------------------8
(e) 無機黏土礦物-----------------------------------------------------------8
1.3 研究目的------------------------------------------------------------------------9

第二章 理論基礎---------------------------------------------------------------------10
2.1 蒙脫石結構與性質-----------------------------------------------------------10
2.2 烷基胺離子改質蒙脫石-----------------------------------------------------12
2.3 報導基因-----------------------------------------------------------------------14

第三章 實驗方法---------------------------------------------------------------------16
3.1 實驗儀器-----------------------------------------------------------------------16
3.2 實驗藥品-----------------------------------------------------------------------17
3.3 實驗方法與流程--------------------------------------------------------------18
3.3.1 鈉-蒙脫石之製備-------------------------------------------------------19
3.3.2 HDTMA-蒙脫石之製備-----------------------------------------------20
3.3.3 質體DNA的抽取-------------------------------------------------------21
3.3.4 線性pEGFP-C1之製備------------------------------------------------23
3.3.5 洋菜膠體電泳-----------------------------------------------------------23
3.3.6 線性DNA/HDTMA-蒙脫石複合物之製備----------------------24
3.3.6.1 不同反應時間下製備線性DNA/HDTMA-蒙脫石複合物
-------------------------------------------------------------------25
3.3.6.2 不同重量比例下製備線性DNA/HDTMA-蒙脫石複合物
-------------------------------------------------------------------25
3.3.6.3 不同pH與陽離子交換量下製備質體DNA/HDTMA-蒙脫石複合物-----------------------------------------------------------25
3.3.7 質體DNA/HDTMA-蒙脫石複合物之製備----------------------26
3.3.7.1 不同反應時間下製備質體DNA/HDTMA-蒙脫石複合物
-------------------------------------------------------------------27
3.3.7.2 不同重量比例下製備質體DNA/HDTMA-蒙脫石複合物
-------------------------------------------------------------------27
3.3.7.3 不同pH與陽離子交換量下製備質體DNA/HDTMA-蒙脫
石複合物-----------------------------------------------------------27
3.3.8 DNA/HDTMA-蒙脫石複合物之分析實驗-----------------------28
3.3.9 HDTMA-蒙脫石複合物之MTT測試-------------------------------29
3.3.10 DNA/HDTMA-蒙脫石複合物之轉染實驗---------------------30
3.4 材料分析-----------------------------------------------------------------------31
3.4.1 X光繞射儀分析---------------------------------------------------------31
3.4.2 粒徑分析-----------------------------------------------------------------31
3.4.3 分光光度計分析--------------------------------------------------------32

第四章 結果及討論-----------------------------------------------------------------33
4.1 XRD繞射分析---------------------------------------------------------------33
4.2 粒徑分析-----------------------------------------------------------------------35
4.3 線性pEGFP-C1之製備------------------------------------------------------38
4.4 pEGFP-C1/HDTMA-蒙脫石複合物製備之反應條件---------------38
4.4.1 反應時間之影響--------------------------------------------------------39
4.4.2 不同改質蒙脫石的重量比例之影響--------------------------------41
4.4.3 不同陽離子交換量HDTMA-蒙脫石之影響--------------------42
4.4.4 不同反應的pH值之影響----------------------------------------------44
4.5 MTT測試----------------------------------------------------------------------47
4.6 DNA/HDTMA-蒙脫石複合物之分析----------------------------------48
4.7 DNA/HDTMA-蒙脫石複合物之轉染實驗----------------------------50

第五章 結論--------------------------------------------------------------------------52

第六章 未來展望--------------------------------------------------------------------53

第七章 參考文獻--------------------------------------------------------------------54
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