臺灣博碩士論文加值系統

聚賴胺酸（Poly-L-lysine ; PLL）是生物可降解性的高分子，由於其低毒性之特性已廣泛運用於組織工程。近幾年有關於亞胺（imine）官能基的水解性質已被深入地探討，但目前只有少數相關的研究將亞胺運用於基因載體材料上。本研究中，成功地合成出以聚賴胺酸為主鏈，利用苯甲醛作為連結以亞胺鍵結聚乙烯胺（PEI），在結構側鏈末端懸掛分子量較低的聚乙烯胺（PEI423和PEI600），形成高分枝型態的陽離子型高分子（分別為PLI423和PLI600），並將其運用於非病毒型基因傳遞的載體上。
實驗結果顯示，在酸鹼滴定試驗指出PLI系列之陽離子型高分子有不錯的緩衝能力；由於PLI本身易水解的性質而於細胞實驗中呈現低毒性的表現。此外，粒徑測試之結果證實，PLI423和PLI600皆能輕易地與質體DNA自組裝成奈米級的複合體，大小在200奈米左右，且複合物顆粒表面帶有正電荷（~ + 25 mV）。在體外基因轉染試驗方面，PLI423和PLI600與原始的聚賴胺酸和低分子量的聚乙烯胺相比較，高分子PLI之轉染效率高出了10倍，並且利用螢光顯微鏡的影像以及流式細胞儀分析，再次證實高分子PLI有很高的轉染效率，且基因傳遞效果比目前市售的高分子量之聚乙烯胺（PEI25K）更佳。簡單來說，本研究提供了一個新的製備方式去合成低毒性且具高轉染效率的陽離子型高分子，由於傳遞基因之高效率和低細胞毒性及生物可分解性，可望在未來運用於多種基因缺陷之疾病。

Over the past few years, a number of studies have been conducted to focus on the hydrolysis property of imine groups. Research on cationic polymers contain imine groups in related to their capability of gene delivery has not yet been investigated. In this research, a new series of poly(L-lysine) – grafted – polyethylenimine（PLI）were successfully synthesized. The grafted polymer contains poly(L-lysine) as a backbone with and low-molecular-weight polyethylenimine（LMW-PEI）in the side chains via imine linkages, was examined for their potentials as non-viral gene vectors which deliver plasmid DNA into cells in vitro.
The electrophoretic mobility on agarose gels, dynamic light scattering and zeta potential analyses of polymer∕DNA complexes confirmed that PLIs were able to self-assemble with plasmid DNA to form nano-scaled（around 200 nm）and positively charged complexes（~ + 25 mV）. PLIs hardly showed any cytotoxicity on COS-7 cells duo to their biodegradability and biocompatibility. Most importantly, in vitro transfection results showed that PLIs displayed a far better transfection efficiency in comparison to LMW-PEI and poly(L-lysine). Moreover gene delivery results even better than the commercially available high-molecular-weight polyethylenimine (PEI25K). Therefore, these new cationic polymers with imines having low cytotoxicity and high transfection efficiency may be applied as safe and efficient non-viral gene delivery vectors.

謝誌
中文摘要
英文摘要
目錄
圖目錄
表目錄
第一章 文獻回顧
1-1 基因治療的起源與歷史
1-2 基因治療的原理與功用
1-3 基因傳遞系
1-3-1 物理性基因傳遞方式
1-3-2 化學性基因傳遞方法
1-3-3 基因載體傳遞法
1-3-4 非病毒性載體
1-4 陽離子型高分子載體的轉染機制
第二章 研究動機與目的
2-1 聚賴胺酸（Poly-L-lysine；PLL）
2-2 聚乙烯胺（Polyethylenimine；PEI）
2-3 聚賴胺酸嫁接聚乙烯胺之設計
第三章 實驗方法
3-1 實驗藥品
3-2 實驗儀器
3-3 實驗步驟
3-3-1 Benzoic aldehyde-PLL（1）之合成
3-3-2 PLI423 之合成
3-3-3 PLI600 之合成
3-3-4 高分子結構鑑定與分析
3-3-5 PLI之水解測試
3-3-6 PLI之酸鹼滴定（緩衝能力）分析
3-3-7 質體 DNA 之製備
3-3-8 PLI∕DNA複合體之製備
3-3-9 PLI∕DNA複合體之凝膠電泳分析
3-3-10 PLI∕DNA複合體水解後之凝膠電泳分析
3-3-11 PLI∕DNA複合體之粒徑與界面電位分析
3-3-12 細胞培養
3-3-13 細胞毒性測試（XTT assay）
3-3-14 體外基因轉染試驗
3-3-15 流式細胞儀分析
第四章 結果與討論
4-1 Benzoic aldehyde-PLL（1）之合成與鑑定
4-2 PLI之合成與鑑定
4-3 GPC 分子量鑑定
4-4 PLI之水解測試
4-5 PLI之緩衝能力分析
4-6 PLI/DNA複合體之之凝膠電泳分析
4-7 PLI/DNA複合體水解後之凝膠電泳分析
4-8 PLI/DNA複合體之粒徑與界面電位分析
4-9 PLI/DNA複合體之細胞毒性試驗（XTT assay）
4-10 PLI/DNA複合體之體外基因轉染試驗
4-10-1 質體 DNA 為pCMV-β-gal
4-10-2 質體 DNA 為pEGFP-C2
第五章 結論
第六章 參考文獻

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