引入Poly(γ-glutamic acid)於Chitosan/siRNA奈米微粒對細胞胞飲及基因抑制之影響

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研究生：	廖子嫻 Lian, Zi-Xian
論文名稱：	引入Poly(γ-glutamic acid)於Chitosan/siRNA奈米微粒對細胞胞飲及基因抑制之影響 Effects of Incorporation of Poly(γ-glutamic acid) in Chitosan/siRNA Nanoparticles on Cellular Uptake and Gene Silencing
指導教授：	宋信文 Sung, Hsing-Wen
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2009
畢業學年度：	97
語文別：	中文
論文頁數：	56
中文關鍵詞：	幾丁聚醣、聚麩胺酸、核醣核酸
外文關鍵詞：	Chitosan, Poly(γ-glutamic acid), siRNA
相關次數：	點閱：3 下載：0
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摘要
核醣核酸干擾(RNA interference; RNAi)為近年發展的技術，其對於基因抑制方面具有高效性和專一性，為研究基因功能的重要工具之ㄧ。這項新技術可以運用來治療各種疾病和癌症，進而達到抑制該癌症細胞生長或是關閉某些不良基因表現。目前有很多傳遞siRNA之方法，其中非病毒型基因載體擁有許多優點，例如低免疫性和低致病性。這些優點對於傳遞基因到生物體有很好的安全考量。本研究將利用[Poly(γ-glutamic acid)](γ-PGA)引入至Chitosan(CS)/siRNA奈米微粒之組成配方中，探討其改善siRNA傳遞效率和抑制特定基因的效果。實驗結果得知引入γ-PGA之奈米微粒具有穩定包覆siRNA之能力，且其無論在粒徑大小或表面電荷的數值分布上皆較無γ-PGA之奈米微粒來的均一。透過電泳分析及流式細胞儀分析數據顯示，引入γ-PGA之奈米微粒可穩定包覆siRNA及增加胞飲效率。而以冷光儀分析冷光蛋白基因抑制效果可知引入γ-PGA後之CS奈米微粒，對抑制基因表現亦有提升的效果，可使冷光蛋白基因之抑制效率提升18-30%；此外，亦可由共軛焦顯微鏡發現綠色螢光蛋白被抑制的效果，隨著γ-PGA的引入而有提升現象。以上結果證實了，引入γ-PGA所形成之CS奈米微粒可以做為攜帶siRNA之載體且更有效率。

目錄
摘要    I
目錄    II
圖目錄    V
表目錄    VI

第一章 前言    1
1 核醣核酸干擾(RNA interference; RNAi)之發展    1
1.1 核醣核酸干擾之萌芽    1
1.2 核醣核酸干擾之機制    2
1.3 核醣核酸干擾之醫療應用    4
2 核醣核酸之傳遞方式與系統    5
3 幾丁聚醣 (Chitosan; CS)    7
4 聚麩胺酸(γ-poly-glutamic acid; γ-PGA)    8
5 研究動機與目的    8
第二章 CS/siRNA 奈米微粒之最佳化實驗    10
1 研究目的    10
2 材料與方法    10
2.1 低分子量之幾丁聚醣製備    10
2.2 siRNA種類    12
2.3 不同電荷比之奈米微粒製備及電泳分析    12
2.3.1 不同電荷比之CS/siRNA奈米微粒製備    12
2.3.2 電泳分析    12
2.4 細胞培養    13
2.5 奈米微粒之轉染    13
2.5.1 奈米微粒對細胞之毒性分析    14
2.5.2 細胞對奈米微粒之胞飲效率    14
2.6 共軛焦螢光顯微鏡之影像分析    15
3 實驗結果與討論    15
3.1 低分子量幾丁聚醣    15
3.2 CS/siRNA奈米微粒之電泳分析結果    16
3.3 奈米微粒轉染實驗結果    17
3.3.1 細胞毒性分析    17
3.3.2 細胞胞飲效率    18
3.4 共軛焦顯微鏡之影像分析    18
4 結論    20
第三章 CS/siRNA奈米微粒抑制基因表現    21
1 研究目的    21
2 材料與方法    21
2.1 奈米微粒之粒徑及電荷    21
2.2 表現綠色螢光蛋白及冷光蛋白之人類纖維母細胞株HT-1080之建立    21
2.3 CS/siRNA奈米微粒抑制基因表現之時間趨勢實驗    23
2.4 不同轉染時間下奈米微粒對抑制冷光蛋白基因表現之效率分析實驗    23
2.5 共軛焦螢光顯微鏡之影像分析    24
3 實驗結果與討論    24
3.1 奈米微粒之粒徑大小及表面電荷分佈    24
3.2 奈米微粒抑制基因表現之時間趨勢結果    25
3.3 不同轉染時間下之基因受抑制表現結果    26
3.4 共軛焦螢光顯微鏡之影像分析    28
4 結論    30
第四章 引入聚麩胺酸(γ-PGA)於奈米微粒之最佳化    31
1 研究目的    31
2 材料與方法    31
2.1 低分子量聚麩胺酸(γ-PGA)之製備    31
2.2不同電荷比之奈米微粒製備及其特性分析    32
2.2.1 不同電荷比之奈米微粒製備    32
2.2.2 電泳分析    32
2.2.3 奈米微粒之粒徑大小及表面電荷    33
2.3 細胞培養    33
2.4 不同電荷比之奈米微粒與轉染時間差異對細胞之影響    33
2.4.1 胞飲效率之分析    33
2.4.2 基因受抑制表現之效率分析    34
2.5 不同電荷比之奈米微粒對細胞毒性之分析    35
2.6 共軛焦螢光顯微鏡之影像分析    35
3 實驗結果與討論    36
3.1 不同電荷比之奈米微粒特性    36
3.1.1 電泳分析    36
3.1.2 粒徑大小及表面電荷    37
3.2 不同電荷比之奈米微粒及轉染時間差異對細胞之影響    38
3.2.1 胞飲效率分析結果    38
3.2.2 基因受抑制表現之效率分析結果    40
3.3 不同電荷比奈米微粒之毒性分析結果    42
4 結論    44
第五章 引入γ-PGA之奈米微粒抑制基因表現之效率    45
1 研究目的    45
2 材料與方法    45
2.1 添加γ-PGA之奈米微粒抑制基因表現之時間趨勢實驗    45
2.2 共軛焦螢光顯微鏡之影像分析    45
3 實驗結果與討論    46
3.1 添加γ-PGA之奈米微粒抑制基因表現之時間趨勢結果    46
3.2 共軛焦顯微鏡之影像分析    47
4 結論    49
第六章 參考文獻    51

                                

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