臺灣博碩士論文加值系統

基因疫苗是目前疫苗發展的主流，而一種新型的基因傳遞系統，以非病毒的方式攜帶抗原蛋白之基因片段進入宿主體內表達，並強烈刺激黏膜免疫及全身免疫反應，以對抗腸道致病菌。本實驗使用一個由質體DNA、雙硫鍵低分子直鏈聚乙烯亞胺(disulfide-crosslinked low molecular linear　polyetheleneimine, CLPEI)、以及玻尿酸(hyaluronic acid, HA)所組成的三元複合物（DNA-CLPEI-HA），是一種在細胞培養系統中表現出較高的基因轉染效率和低毒性的複合物。實驗中利用電子顯微鏡觀察DNA-CLPEI-HA複合物的物理特性、以DNA電泳以及限制酵素處理的方式測試口服基因傳遞系統包覆DNA的穩定性、在腸道上皮細胞的細胞培養系統中細胞轉染效率、細胞存活率、體外腸道循環以及體內接踵的小鼠實驗。要確認表達所攜帶綠色螢光蛋白(green fluorescence protein, GFP)的黏膜及全身性免疫反應，將取決於血清中的抗GFP的IgG抗體量以及GFP特異性的細胞免疫反應。實驗結果證實在DNA與CLPEI及HA結合後，會形成直徑300~400nm左右的顆粒大小，而且複合物在混和比例N/P比值為100時可以有效保護DNA不被限制酵素切除，在細胞實驗中有良好的轉染效率以及低細胞毒性，但在動物實驗中複合物仍然會對腸道造成些微的損傷。在這項研究中所開發的口服基因傳遞系統，對於用來對抗腸道致病菌及食入和排泄物途徑傳染的病原體的口服疫苗，將成為一個重要的發展平台。

A gene delivery system carrying the gene segment of the antigen protein into the host body to express in non-viral way to stimulate mucosal and systemic immune responses has been developed. The current study used the ternary complex（DNA-CLPEI-HA）composed of plasmid DNA, disulfide-crosslinked low molecular linear polyetheleneimine(CLPEI) ,and hyaluronic acid (HA), which exhibited high gene transfection efficiency and low toxicity in cell culture systems. The examination by electron microscopes showed the physical properties of DNA-CLPEI-HA complex. The stability of DNA in the complex after oral delivery was determined by DNA electrophoresis and the digestions of restriction enzyme. The transfection efficiency of green fluorescence protein (GFP) gene delivered by DNA-CLPEI-HA complex in the intestinal epithelial cells in cell culture systems and the cell viability of the complex were examined in vitro. The expression of GFP and the immune responses induced in mice was evaluated by the levels of anti-GFP IgG antibody. The DNA-CLPEI-HA complex had a particle size of about 300 ~ 400nm. The DNA binding with CLPEI and HA at the mixing ratio N / P ratio 100 can be effectively protected from the digestion of restriction enzymes. Good transfection efficiency and low cytotoxicity were observed in cell culture system. The complex caused mild to no damage to the intestine in mice ingested orally the complex. Thus, the oral gene delivery system developed in this study had potentials to become a platform for oral vaccines used to fight intestinal pathogens.

摘要 I
Abstract II
目錄 III
圖表目錄 V
第一章　緒論 1
第二章　文獻回顧 2
2.1 基因疫苗的特色 2
2.2 DNA疫苗的發展 2
2.3 攜帶DNA的複合物 3
2.3.1 PEI之特性 4
2.3.2 玻尿酸之特性 4
2.3.3 三元複合物DPH 4
2.3.4 PEI的佐劑特性 5
2.4 腸道黏膜免疫反應的原理 6
2.5 口服疫苗的特色 6
第三章　材料與方法 9
3.1 材料 9
3.1.1 藥品與試劑 9
3.1.2 細胞株 9
3.2 方法 11
3.2.1 實驗流程圖 11
3.2.2 質體的備製 11
3.2.3 DNA-CLPEI-HA複合物合成 12
3.2.4 DNA-PEI-HA複合物包覆測試 13
3.2.5 細胞轉染實驗 13
3.2.6 細胞毒性測試 14
3.2.7掃描式電子顯微鏡 14
3.2.8穿透式電子顯微鏡及樣品染色 15
3.2.9 小鼠體外腸環套測試 15
3.2.10 小鼠體內口服給藥測試 16
3.2.11 小鼠產生之綠色螢光抗體測試 16
第四章　研究結果 18
4.1DNA-CLPEI-HA複合物的大小 18
4.2 DNA-CLPEI-HA複合物包覆DNA的效果 18
4.3 細胞毒性測試 18
4.4 細胞轉染實驗 19
4.5 測試不同溶液對於小鼠腸道之毒性 19
4.6 測試小鼠經由口服給藥後體內GFP抗體 19
第五章 討論 34
5.1 DNA-CLPEI-HA複合物的大小 34
5.2 DNA-CLPEI-HA複合物包覆DNA的效果 35
5.3 細胞毒性測試 35
5.4細胞轉染實驗 35
5.5測試不同溶液對於小鼠腸道之毒性 36
5.6測試小鼠經由口服給藥後體內GFP抗體 36
第六章 補充資料 37
6.1 DNA-CLPEI-HA複合物包覆效率測試 37
6.2 DNA-CLPEI-HA複合物細胞毒性測試 37
第六章　參考文獻 43


圖表目錄

圖 一、掃描式電子顯微鏡觀察PEI之結果。 26
圖 二、穿透式顯微鏡觀察結果。 28
圖 三、凝膠電泳測試結果。 29
圖 四、MTS細胞毒性測試結果。 30
圖 五、細胞轉染實驗之結果。 31
圖 六、組織病理切片圖顯示不同溶液對於小鼠腸道之毒性。 32
圖 七、測試小鼠血清中抗體結果。 33

補充圖 一、測試複合物在模擬胃液及模擬腸液處理下的包覆結果。 39
補充圖 二、LDH細胞毒性測試結果。 40
補充圖 三、以239T細胞表達GFP之結果 41
補充圖 四、以Caco-2細胞轉染表達GFP之結果。 42

表 一、MTS、LDH細胞毒性測試各組別用量 20
表 二、體外腸循環分析 23
表 三、口服體內實驗病變評估 24
表 四、不同實驗中的DNA使用量及濃度 25

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