臺灣博碩士論文加值系統

本研究主要目的為設計及合成低毒性之生物可分解性的聚胺基甲酸酯高分子載體，而且將能促使細胞膜不穩定之病毒胜肽鏈進行化學鍵結，利用helix的胜肽鏈促使內質體膜的不穩定，藉此提高基因輸送的效果。
實驗內容包含了探討聚胺基甲酸酯高分子與DNA所形成的複合體之物理化學性質，如：電泳膠片分析、粒徑（DLS）、在仿生體液（HEPES）中裂解速率、緩衝能力、對哺乳類動物細胞毒性、及哺乳類動物細胞的轉染效率及其細胞內吞作用的路徑。
我們成它合成出在高分子主鏈及側鏈上帶有胺基的新型聚胺基甲酸酯(PU)系統，並在側鏈上進行置換不同的二胺類，包括了一級胺，三級胺等二胺類。來探討其側鏈種類、鏈長對轉染效率的影響。研究結果發現，此聚胺基甲酸酯在水溶液中具有非常好的溶解度，也可以快速的裂解，因此沒有明顯細胞毒性，其中PU-PA-1、 PU-PA-3、PU-PA-1-peptide適當的重量比之下，可以有效地保護質體DNA，並且能夠在48小時內成它a轉染哺乳類動物細胞，加入病毒胜肽鍵的PU-PA-1-peptide對於COS-7細胞其最佳轉染效率能高於PDMAEMA的表現。

The goal of this project is to design and synthesize biodegradable cationic polyurethanes with low Cytotoxicity. A membrane-disrupting peptide derived from the influenza virus was covalently linked to the cationic polyurethane to increase the transfection efficiency of PU-based gene delivery systems.
The cationic polyurethane/plasmid DNA complexes were characterized for their physical and chemical properties，by examining their electrophoresis mobility on agarose gel，particle size (DLS)，the hydrolytic degradation， buffering capacity，cytotoxicity，transfection efficiency，and possible endocytosis pathways.
In our study, a series of cationic polyurethane with various structures in side chain has been systemic investigated. These side chains (primary amine or tertiary amine) would carry positive charges so that the relations of different amines and length of side chains with transfection activity of polyurethane can be studied. Results show that these cationic cationic polyurethanes are able to transfect mammalian cells with advantageous properties of low cytotoxicity, water solubility and biodegradability in water after 48 hours.

總目錄
總目錄 -------------------------------------------------------------------- III
中文摘要---------------------------------------------------------------------I
英文摘要-------------------------------------------------------------------- II
圖表目錄 --------------------------------------------------------------------VI
縮寫表 ----------------------------------------------------------------------X
第一章 緒論 ---------------------------------------------------------------- 1
1.1、 引言 -------------------------------------------------------------------1
1.1.1、 基因治療背景與發展--------------------------------------------1
1.2、基因遞送系統------------------------------------------------------------5
1.2.1、 病毒性載體 -----------------------------------------------------5
1.2.2、 非病毒性載體 ---------------------------------------------------- 9
1.3、基因轉殖的機制與障礙因素 ---------------------------------------- 19
1.3.1、標的細胞之再吸收 --------------------------------------------- 19
1.3.2、內質體之釋放 --------------------------------------------------- 20
1.3.3、在細胞質中安定性與細胞核輸入 --------------------------- 21
1.4、生醫材料 ------------------------------------------------------------- 21
1.4.1、生物可分解性高分子 ------------------------------------------ 21
1.4.2、異氰酸酯化學反應 --------------------------------------------- 29
1.5、質體DNA --------------------------------------------------------------- 32
1.6、研究動機 --------------------------------------------------------------34
第二章 實驗材料------------------------------------------------------------ 37
2.1、實驗儀器 ------------------------------------------------------------32
2.2、實驗藥品------------------------------------------------------------- 39
2.3、實驗方法--------------------------------------------------------------41
2.3.1、質體DNA的製備 ---------------------------------------------- 41
2.3.2、細胞株 ------------------------------------------------------------ 45
2.3.3、實驗方法流程圖-------------------------------------------------- 48
2.3.4、電泳實驗 --------------------------------------------------------- 57
2.3.5、測定高分子分子量 --------------------------------------------- 60
2.3.6、測定粒徑大小 --------------------------------------------------- 61
2.3.7、測定緩衝能力 --------------------------------------------------- 62
2.3.8、細胞毒性分析 --------------------------------------------------- 63
2.3.9、細胞轉染實驗 --------------------------------------------------- 65
2.3.10、細胞內吞的路徑分析-------------------------------------69
第三章 結果與討論 ------------------------------------------------------- 72
3.1、聚胺基甲酸酯高分子的分子量探討 -------------------------------- 72
3.2、高分子與質體DNA形成複合體的能力 --------------------------- 73
3.2.1、 PU-1、PA、PU-PA、PU-PA-1高分子對質體DNA進行包覆的效---------------73
3.2.2、PU-3、PU-PA-3高分子對質體DNA進行包覆的效率----- 75
3.2.3、PU-1-peptide、PU-PA-1-peptide高分子對質體DNA進行包覆的效率------- 77
3.2.4、PU-PA-1，PU-PA-1-peptide高分子的水解能力-------------- 78
3.3、粒徑大小分析與測定 --------------------------------------------------- 81
3.4、高分子的酸鹼滴定—探討高分子的緩衝能力 -------------------- 86
3.5、高分子對細胞毒性大小測試 ----------------------------------------- 90
3.6、細胞轉染實驗--------------------------------------------------------- 94
3.7、探討細胞內吞作用的路徑實驗--------------------------------------- 100
第四章 結論 ------------------------------------------------------------- 103
4.1、聚胺基甲酸酯結構之特性 ------------------------------------- 103
4.2、 聚胺基甲酸酯之分子量 --------------------------------------- 103
4.3、聚胺基甲酸酯之緩衝能力 ------------------------------------- 104
4.4、聚胺基甲酸酯之細胞毒性 ------------------------------------- 106
4.5、總結 ------------------------------------------------------------ 106
第五章 結構圖譜鑑定 ---------------------------------------------------- 108
參考文獻 ------------------------------------------------------------------ 118

圖表目錄
表1.1、適用於基因治療的一些遺傳性疾病-------------------------------- 3
表1.2、病毒性載體內的片段的胜肽------------------------------------------ 7
圖1.3、通常基因轉染進入細胞的方法------------------------------------- 10
圖1.4、微注射(microinjection)的轉染方式--------------------------------- 11
圖1.5、微脂粒與細胞膜間的相互作用------------------------------------- 14
圖1.6、目前常見的四種陽離子型高分子系統---------------------------- 16
圖1.7、質子海綿體效應------------------------------------------------------- 18
圖1.8、陽離子型載體的轉染機制------------------------------------------- 19
圖1.9、pCMV-LacZ 質體 DNA的圖譜------------------------------------ 34
圖1.10、胜肽鏈在正常pH值的狀態下會因電荷的排斥形成隨機的捲曲狀態，然而在較低的pH值下，由於質子化的反應使的胜肽形成兩極化的?陰蛣硎c------------------ 35
表 2.1、Luria Broth 的組成 ------------------------------------------------- 41
表 2.2、QIAGEN plasmid mega kit -------------------------------------- 44
圖 2.1、COS-7 細胞生長情形 -------------------------------------------- 45
圖2.2、聚胺基甲酸酯高分子（PU）的合成 --------------------------- 50
圖2-3、側鏈帶有一級胺類的PU-1、PU-PA-1 ----------------------------- 52
圖2.4、側鏈帶有三級胺類的PU-3、PU-PA-3 ----------------------------- 54
圖2.5、含胺基酸化合物化合物Inf7的PU-1-peptide、PU-PA-1-peptide ------------ 56
圖2.6、螢光化合物 ethidiumbromide 的化學結構式------------------ 58
圖2.7、XTT的化學結構式 ------------------------------------------------- 63
圖2.8、XTT在細胞中反應形成formazan鹽類---------------------------- 64
表 2.3、XTT試劑的組成------------------------------------------------------ 65
圖2.9、細胞胞飲的三種抑制路徑------------------------------------------- 69
表 3.1、聚胺基甲酸酯分子量及多分佈指數之數據 -------------------- 72
圖3.1、PU-1、PA、PU-PA、PU-PA-1高分子與質體DNA形成複合體的包覆能力---------- 73
圖3.2、PU-3、PU-PA-3高分子與質體DNA形成複合體的包覆能力--------------------- 75
圖3.3、PU-1-peptide、PU-PA-1-peptide高分子與質體DNA形成複合體的包覆能力----- 77
圖3.4、PU-PA-1、PU-PA-1-peptide高分子與質體DNA形成複合體的水解能力------- 78
圖3.5、PU-1、PU-PA-1、PA、PU-PA、PU-3、PU-PA-3在不同濃度比例下與DNA形成複合體的粒徑大小分佈------------------- 81
圖3.6、PU-1、PU-1-peptide在不同濃度比例下與DNA形成複合體的粒徑大小分佈----- 82
圖3.7、PU-PA-1、PU-PA-1-peptide在不同濃度比例下與DNA形成複合體的粒徑大小分佈 84
圖3.8、不同側鏈的陽離子型高分子聚胺基甲酸酯在酸滴定下
的緩衝能力曲線-------------------------------------------------------- 86
圖3.9、PU-1、PU-1-peptide的陽離子型高分子聚胺基甲酸酯在酸滴
定下的緩衝能力曲線---------------------------------------------- 88
圖3.10、PU-PA-1、PU-PA-1-peptide的陽離子型高分子聚胺基甲
酸酯在酸滴定下的緩衝能力曲線------------------------------- 89
圖3.11、高分子細胞毒性分析針對PU-1、 PU-PA-1、PU-PA-3、PU-PA-3在不同濃度比例下，其對細胞的毒性大小測試-------------- 90
圖3.12、高分子細胞毒性分析針對PU-1、 PU-1-peptide、 PU-PA-1、PU-PA-1-peptide在不同濃度比例下，其對細胞的毒性大小測試--------------------------------- 91
圖3.13、PU-1、PU-PA-1、PU-3、PU-PA-3等PU系統以不同的濃度比例進行細胞轉染實驗 94
圖3.14、PU-1、PU- 1-peptide以不同的濃度比例進行細胞轉染實驗------------- 97
圖3.15、PU-PA-1、PU- PA-1-peptide以不同的濃度比例進行細胞轉染實驗----------- 98
圖3.16、使用X-gal染色法觀察受到 PEI 、PDMAEMA、PU-PA-1-peptide轉染的細胞數目 99
圖3.17、PEI 、PDMAEMA、PU-PA-1-peptide轉染的COS-7細胞，經過X-gal染色後的情形-------------------------------------- 100
圖3.18、 PU-PA-1、PU-PA-3、PU-PA-1-peptide轉染效率圖----------- 100

縮寫表

PU Polyurethane
PA Poly-L-arginine
PAA Poly(amido amine)
PLL Poly-L-Lysine
PEI Polyethylenimine
PDMAEMA Poly(N,N-dimethylaminoethyl methacrylate)
PBS Phosphate buffer saline
SPDP 3-(2-Pyridyldithio)propionic acid
N-hydroxysuccinimide ester
Inf7 “N”-GLF EAI EGFI ENGW EGMI DGWYGC-NH2 - “C”
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetates
FTIR Fourier Transform Infrared Spectroscopy
NMR Nuclear Magnetic Resonance
GPC Gel permeation chromatography

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