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研究生: 吳韶淳
Shao-Chun, Wu
論文名稱: 聚乙烯亞胺對於陽離子微脂粒基因載體系統的影響
The Effect of Polyethylenimine on the Cationic Liposome Gene Delivery System
指導教授: 朱一民博士
I-min, Chu
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: 2007
畢業學年度: 95
語文別: 中文
論文頁數: 52
中文關鍵詞: 基因載體陽離子微脂粒聚乙烯亞胺
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  • 近年來由於基因療法的興起,使得基因載體成為重要的研究目標。病毒型載體雖然可以得到高轉染率,但是會衍生出病毒變異而產生副作用等疑慮。目前較被廣泛研究的非病毒型載體轉殖方法為微脂粒系統,雖然比較安全,轉染率卻不若病毒型載體高。本文中使用的陽離子微脂粒系統是由帶正電荷的脂質DC-Chol和中性脂質DOPE製備而成。DC-Chol能和帶負電荷的DNA藉由靜電力作用形成複合體,而DOPE有助於轉染過程中DNA的釋放。
    聚乙烯亞胺( PEI, polyethylenimine )為帶正電荷的高分子,可幫助內質體破裂而釋放出包覆的DNA。本研究將製備好的陽離子微脂粒和PEI、DNA共置形成複合體,在中國倉鼠卵巢細胞( CHO-K1 )中進行轉染實驗。實驗結果發現相較於原本的陽離子微脂粒系統,添加PEI的新系統的確能夠將轉染率提升至原先的6倍;PEI添加適當比例,也能將細胞存活率維持在60%以上;另外,PEI的存在還能降低微脂粒系統對血清的敏感度,使本系統未來在動物實驗更具發展性。
    由上述結果可知,聚乙烯亞胺和陽離子微脂粒共同作用的基因傳遞系統值得深入研究探索,未來也可利用不同正電荷脂質來研究PEI的效果,或是對PEI進行改質以標的特定轉染細胞,甚至進入動物實驗來進一步發展完整的系統。


    Recently, because of the general attention to the gene therapy, gene carriers become an important issue. The transfection efficiency of viral vectors is high, but there are some considerable problems such as the mutation. So far, the most well developed nonviral vectors are liposomes. Although liposomes are known as safer carriers, their transfection efficiency remains low. We used cationic liposomes composed of a cationic lipid DC-Chol and a neutral lipid DOPE. DC-Chol could form complexes with DNA through the electrostatic force, and DOPE helped the release of DNA.
    Polyethylenimine (PEI) is a positively charged polymer. It induces the endosome to break and the lipoplexes could escape more easily. In this research, we co-placed the cationic liposome, PEI and DNA to form complexes, and then went on the transfection in CHO-K1 cells. The results showed that PEI can enhance 6-fold in the transfection efficiency; the cell viability remained higher than 60% with the appropriate amount of PEI; moreover, the existence of PEI could lower the serum sensitivity of the gene delivery system, and it was beneficial to improve the applicability on the in vivo experiments.
    Therefore, the gene delivery system composed of PEI and cationic liposome was worthy of intensive research. We can use different cationic lipid to explore the general effects of PEI, or modify PEI to target the specific cells, and finally we can proceed with the in vivo experiment to develop a complete gene delivery system.

    摘要 I ABSTRACT II 目錄 III 圖目錄 VI 表目錄 VIII 第一章、文獻回顧 1 1.1 基因療法和載體 1 1.2 正電荷高分子載體 2 1.3 微脂粒載體 3 1.3.1 構造 3 1.3.2 和細胞間作用方式 5 1.3.3 正電荷微脂粒 7 1.3.4 正電荷微脂粒系統的轉染作用機制 10 1.3.5 正電荷微脂粒系統的限制與瓶頸 12 1.4 流式細胞儀 13 1.5 細胞存活率測定 15 第二章、研究動機與目的 16 第三章、實驗藥品、儀器與方法 17 3.1 實驗藥品 17 3.2 實驗儀器和耗材 18 3.3實驗步驟 19 3.3.1合成正電荷脂質DC-Chol 19 3.3.2 製備微脂粒 20 3.3.3 抽取質體DNA 21 3.3.4 Lipoplexes的製備 22 3.3.5 細胞轉染實驗 24 3.3.6 流式細胞儀 25 3.3.7 細胞存活率測定 25 第四章、結果與討論 27 4.1 DC-CHOL的合成 27 4.2 脂質比例 28 4.3 質體DNA濃度 29 4.4 PEI濃度 32 4.4 決定轉染率的變因 37 4.5 血清敏感度 38 4.6 討論 42 第五章、未來展望 45 第六章、參考文獻 47

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