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研究生:陳建州
研究生(外文):Jian-Zhou Chen
論文名稱:鹼基拓印甲殼素磁性複合奈米粒子的製備及其在基因轉染及細胞凋亡的應用
論文名稱(外文):Preparation and Characterization of Magnetic Nucleobases Imprinted Chitosan Composite Nanoparticles and Their Applications in Gene Transfection and Cellular Apoptosis
指導教授:林宏殷
指導教授(外文):Hung-Yin Lin
學位類別:碩士
校院名稱:國立高雄大學
系所名稱:化學工程及材料工程學系碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:115
中文關鍵詞:磁性基因載體鹼基分子拓印甲殼素基因轉譯端粒體細胞凋亡
外文關鍵詞:Magnetic Gene VectorsNucleobasesMolecular Imprinted ChitosantelomereCellular Apoptosis
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本研究結合分子拓印技術、甲殼素及磁性奈米粒子,製備鹼基分子拓印甲殼素磁性奈米粒子作為基因載體,並應用於轉染及誘導癌細胞凋亡。以相變化的方式在水相進行製備,保留材料原始特性並維持系統單純,減少成本、耗時同時避免額外的細胞毒性。將0.1g/L鹼基分子拓印於0.01wt%、分子量370k之甲殼素製備複合奈米粒子,利用高效液相層析儀(HPLC)、穿透式電子顯微鏡(TEM)、粒徑分析儀及拉曼光譜儀等進行複合粒子性質分析後,對HEK293及HepG2細胞進行給藥,發現複合甲殼素後可有效減少磁性粉體約35~50%的細胞毒性,利用胞嘧啶拓印甲殼素複合粒子(Cytosine Imprinted Polymers, CIPs)吸附綠螢光蛋白基因,同時外加磁場對HEK293細胞進行轉染,效率可達市售載體的五成以上;另外以胸腺嘧啶拓印甲殼素複合粒子(Thymine Imprinted Polymers, TIPs)阻礙端粒體行為,結果在給藥後發現癌細胞分裂明顯趨緩,若在表面吸附短片段人工端可成功誘導癌細胞凋亡。
This study combines molecular imprinting technology, chitosan and magnetic nanoparticles to prepare magnetic nucleobases imprinted chitosan composite nanoparticles (MNIC-NPs) as gene vectors. During the experiments, we imprint adenine, cytosine and thymine on chitosan and use them for gene transfection and induction of cancer cells apoptosis. Using phase transformation to prepare MNIC-NPs in aqueous solution can maintain the nature properties of chitosan and simplify the system, which also can decrease cost and prevent excess cytotoxicity. Imprinting 0.1g/L nucleobases on 0.01wt% chitosan with 370k M.W., then analyzing properties by high performance liquid chromatography, transmission electron microscopy, particle size analyzer and Raman spectrum. Chitosan can reduce 35~50% cytotoxicity of magnetic particles which dose to HEK293 and HepG2 cells. Adsorb green fluorescent protein gene (pEGFP) with cytosine imprinted polymers (CIPs) and transfect into HEK293 cells by magnetic field, the efficiency can surpass 50% which compare with commercial vectors (Lipofection 2000). After dosing thymine imprinted polymers (TIPs, which are expected to block the function of telomeres) into HepG2 cells, the growth rate slowing down obviously. Furthermore, rebinding artificially short telomeres on TIPs before dosing can successfully induce apoptosis of HepG2.
致謝 I
目錄 V
表目錄 IX
圖目錄 X
摘要 1
ABSTRACT 2
第一章 前言 3
1.1研究背景 3
1.2實驗動機 4
1.3論文概要 5
第二章 文獻回顧 6
2.1基因載體 6
2.1.1基因載體的起源 6
2.1.2病毒載體 9
2.1.3非病毒式基因載體 12
2.1.4其他方式及前景 15
2.2分子拓印高分子技術 16
2.2.1緣起 16
2.2.2原理 17
2.2.3拓印孔洞的辨識機制及親合位置 19
2.2.4分子拓印高分子技術之應用 21
2.3甲殼素 23
2.3.1甲殼素基本性質 23
2.3.2藥學上的應用 23
2.3.3甲殼素基因載體 24
2.3.4分子拓印甲殼素的應用 27
2.4端粒體 29
2.4.1發現及功能 29
2.4.2端粒體功能與抗癌 32
第三章 實驗儀器與步驟 34
3.1 實驗藥品與儀器 34
3.1.1實驗藥品 34
3.1.2實驗材料 37
3.1.3 實驗儀器 38
3.2分子拓印磁性奈米粉體 40
3.2.1磁性奈米粉體合成 40
3.2.2分子拓印高分子磁性複合粉體製備 42
3.2.3複合粉體之吸附能力分析 43
3.2.4複合粉體粒子物性分析 45
3.3細胞培養 45
3.3.1緩衝溶液配置 45
3.3.2細胞培養液配製 45
3.4細胞實驗 47
3.4.1細胞毒性實驗 47
3.4.2細胞核酸萃取步驟 47
3.4.3即時定量聚合酶反應器步驟 47
3.4.5誘導啟動HepG2凋亡路徑實驗 48
第四章 結果與討論 49
4.1甲殼素分子量及濃度之影響 49
4.1.1甲殼素濃度對吸附量之影響 49
4.1.2 甲殼素分子量對吸附量之影響 52
4.1.3甲殼素分子量對粒徑之影響 54
4.2鹼基分子拓印甲殼素磁性複合奈米粒子性質分析 56
4.2.1複合微粒顯微影像 56
4.2.2拓印濃度對吸附量之影響 58
4.2.3拓印濃度對複合微粒粒徑之影響 60
4.2.4目標分子的移除 62
4.2.5吸附時間及濃度對吸附量之影響 64
4.2.6分子拓印複合微粒辨識能力 67
4.3分子拓印甲殼素磁性複合奈米微粒之應用 70
4.3.1 CIPs對HEK293細胞生存率之影響 70
4.3.2以CIPs攜帶pEGFP對HEK293進行轉譯 73
4.3.3 TIPs對HepG2細胞生存率之影響 77
4.3.4 TIPs對HepG2給藥後對凋亡基因的影響 79
參考文獻 86
附錄 95
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