臺灣博碩士論文加值系統

在這研究中主要去合成兩種以PU作為主鏈衍生物之陽離子型高分子，並探討相互間轉染之差異。首先透過以PU為主鏈，側鏈選用分子量為423之直鏈型聚乙烯胺，經由胺解反應得到一較短鏈之PUS423，其次透過以PU為主鏈，側鏈選用分子量為423與600，結構分別為直鏈型與團狀型之聚乙烯胺，並利用苯甲醛作為連結以亞胺鍵結聚乙烯胺，得到一較長側鏈之PUI423與PUI600，同時含有imine官能基團。目的為了解PUS423、PUI423與PUI600短鏈與長鏈相互間轉染效率與細胞毒性之差異。並且更進一步比較先前實驗室所合成的PLI423與PLI600轉染效率與細胞毒性之探討。透過PEI25K在體外(vitro)轉染上已被公認做為一個黃金指標，以便評估新型陽離子基因載體傳遞效果的標準。這項研究中我們發現PUI423與PUI600皆能輕易地與質體 DNA 自組裝成奈米級的複合體，粒徑大小小於 200 奈米且複合體表面上帶正電荷。高分子PUI423與PUI600比市售的基因載體材料PEI25K轉染上有更優異的表現與較低細胞毒性。由實驗結果證實了長側鏈轉染效率高於短側鏈，與側鏈轉染效率影響力優越於主鏈的影響力。
關鍵字：非病毒性基因載體、陽離子型高分子、聚胺脂、聚乙烯胺

In this research, two kinds of biodegradable PU derivatives were studied for their differences in transfection efficiency. One of the PU derivatives was successfully synthesized by aminolysis reaction to have polyurethane (PU) as backbone with low molecular weight polyethylenimine (Mw=423) shorter side chains (as PUS423). Another kind was an acid-liable polyurethane-grafted-polyethylenimine (Mw=423 or 600) (as PUI423 or PUI600) were synthesized by making PU as backbone with 4-carboxybenzaldehyde and polyethylenimine in the side chains via imine linkages. For the purpose of understanding the effect of different side chain length on transfection efficiency, the toxicity and the transfection efficiency of PUI423, PUI600 , PUS423 and previous work (PLI423 and PLI600)were compared. Branched PEI25k has been used as a standard reference to compared with our newly designed polymers. This study found that PUI423 and PUI600 were able to bind plasmid DNA and yielded positively charged complexes within a nano-sized range (< 200 nm). The PLI423/DNA and PLI600/DNA complexes were able to transfect COS-7 cell in vitro with better transfection efficiency than other PUS423/DNA and PEI25K/DNA. These results demonstrate that the polymers with longer chain have positive influence and side chain effects better than backbone on transfection efficiency.
Keywords：non-viral vectors、polycations、polyurethane、polyethylenimine

目錄
謝誌 I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 VII
表目錄 X
第一章 緒論 1
1-1 基因治療的起源與發展 1
1-2 基因治療的原理與功用 3
1-3 基因載體治療之方法 5
1-3-1 病毒型載體法(Viral vector) 6
1-3-2 非病毒型載體(Non-viral vector) 9
1-4 陽離子型高分子載體的轉染機制 13
第二章 研究動機與目的 16
2-1 聚氨酯（Polyurethane；PU） 16
2-2 聚氨酯嫁接聚乙烯胺之設計（PUS423,PUI423與PUI600） 17
2-3 聚氨酯與聚賴氨酸分別嫁接聚乙烯胺之轉染探討 21
第三章 實驗方法 23
3-1 實驗藥品 24
3-2 實驗儀器 27
3-3 實驗步驟 29
3-3-1 PU之合成 29
3-3-2 PUS423 之合成 30
3-3-3 PUI423 之合成 31
3-3-4 PUI600 之合成 35
3-3-5 高分子結構鑑定與平均分子量分析 36
3-3-6 PUS423,PUI423與PUI600之水解測試 37
3-3-7 PLI之酸鹼滴定（緩衝能力）分析 37
3-3-8 質體 DNA 之製備與特性 37
3-3-8-1 菌株保存與培養 37
3-3-8-2 質體DNA製備流程 37
3-3-8-3 質體DNA濃度與純度分析 41
3-3-9 PUS(I)∕DNA複合體之凝膠電泳分析 41
3-3-10 PUS(I)∕DNA複合體水解後之凝膠電泳分析 42
3-3-11 PUS(I)∕DNA複合體之粒徑分析 42
3-3-12 細胞培養 43
3-3-12-1 細胞株 43
3-3-12-2 配置細胞培養液 43
3-3-12-3 冷凍細胞活化之流程 44
3-3-12-3 繼代培養 44
3-3-13 細胞存活率試驗（XTT assay） 45
3-3-14 體外基因轉染檢驗(ONPG assay) 46
3-3-15 綠色螢光蛋白基因表現(Flow cytometry analysis) 46
第四章 結果與討論 50
4-1 PU之合成與鑑定 50
4-2 PUS之合成與鑑定 51
4-3 PUI之合成與鑑定 51
4-4 GPC分子量鑑定 55
4-5 PUS(I)之降解能力之探討 59
4-6 PUS(I)之緩衝能力分析 65
4-7 PUS(I)/DNA複合體之凝膠電泳分析 66
4-8 PUS(I)/DNA複合體水解後之凝膠電泳分析 70
4-9 PUS(I)/DNA複合體之粒徑分析 72
4-10 PUS(I)/DNA複合體之細胞存活率試驗（XTT assay） 74
4-11 PUS(I)/DNA複合體之體外基因轉染檢驗(ONPG assay) 77
4-11-1 質體 DNA(pCMV-LacZ)轉染比較 77
4-11-2 質體 DNA (pEGFP-C2)轉染探討 80
第五章 結論 88
第六章 參考文獻 90

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