臺灣博碩士論文加值系統

　　本實驗中，我們評估一種新的口服疫苗輸送系統：胃酸成膠性褐藻膠包覆銀耳多醣修飾微脂粒；結合褐藻膠之高分子聚合物(抵抗胃酸破壞)、微脂粒(疫苗佐劑)輸送系統和銀耳多醣(免疫刺激)特性。我們利用95 % PC之多層球微脂粒包覆106.0 EID50/mL H5N3(LV組)死毒再用銀耳多醣(TFP) 修飾微脂粒表面24小時(LTV組)。單純H5N3病毒 (V組)、LV、 LTV組分別與含有磷酸三鈣之1.5 % alginate混合 (AV、ALV、ALTV組)。
　　將疫苗與胃酸成膠性褐藻酸混合，並進行體外溶離試驗，當胃酸成膠性褐藻酸與模擬胃液接觸時就會形成褐藻膠將微脂粒疫苗包覆起來，而保護微脂粒疫苗不受胃酸破壞，並且在進入腸道時可以調節釋放微脂粒疫苗。而經由單層Caco-2細胞的跨細胞上皮電阻值 ( TEER )實驗顯示，在腸道釋放後之微脂粒疫苗，LV和LTV組都能夠打開Caco-2細胞單層膜間的緊密聯合(分別是77.4%與75.4%比率，兩組沒有顯著差異)。而在TEER的恢復實驗中，可以觀察到LV與LTV組給予Caco-2之後，再給予細胞培養後細胞間緊密聯合可以恢復，細胞也能存活。
動物實驗顯示褐藻膠包覆之疫苗在第二次免疫後3週可以誘發腸道sIgA的產生，雖然在血清中IgG沒有預期中的提高，且血球凝集抑制實驗也顯示沒有足夠的抗體保護效力。但是有可能是因為行Th1免疫途徑而抑制了體液性免疫反應。
　　經由以上的結果顯示，胃酸成膠性銀耳多醣修飾之微脂粒口服疫苗對於提高腸黏膜IgA有一定的能力，而這樣型式的一種疫苗載體再經過材料上的改進，期盼其具有攜帶不易經由口服吸收的疫苗載體的潛力。

In this study, we evaluate a novel delivery system for oral vaccines, TFP-modified liposomes entrapped in acid-induced alginate (ALT) of biodegradable polymers, which is conceived from a combination of the polymer, the lipid–based delivery system and immunostimulant. We utilized liposomes (multilamellar, MLV) of 95 % PC to encapsulate 106.0 EID50/mL inactive H5N3 virus (LV group) and coated with Tremella fuciformis polysaccharide (TFP), purified from hot water extracts of Tremella fuciformis, for 24 hr (LTV group). The virus(V), LV, LTV mixed within 1.5 % alginate with tricalcium phosphate, respectively (AV, ALV and ALTV group).
The vaccine mixed with acid-induced alginate used for in vitro release study. When HCl-induced alginate contact with HCl to forming alginate gel that was more resistant in acidic pH and modulated the release profiles of the encapsulated vaccines in the alkaline pH. Transepithelial electrical resistance (TEER) studies revealed that LV and LTV were able to opening the tight junctions (about 77.4 % and 75.4 % of the initial value, respectively) of Caco-2 cell monolayer by about three times. Recovery studies on the TEER showed that the effect of the LV and LTV vaccines on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all vaccines.
The animal exp. showed that vaccine entrapped in alginate gel induced intestinal sIgA production at 3wk after second administration. although IgG in serum not expected to increase, and the hemagglutination inhibition test also showed that there was insufficient protection of the effectiveness of the antibody. However, there may be because it means Th1 immune suppression of the humoral immune response.
In conclusion, the TFP modified liposomal oral vaccine entrapped in HCl-induced alginate gel for improving the intestinal mucosal IgA have a certain capacity, and this type of a vaccine vector for another material improvement, and this vaccine delivery system may have potential use as a carrier for vaccine that are poorly absorbed after oral administration.

致謝 i
中文摘要 ii
英文摘要 iii

第一章 緒論 1

第二章 文獻回顧 4

§I 家禽流行性感冒病毒之特性 4
•I-1 一般特性 4
•I-2 物理特性 6
•I-3 病毒蛋白特性 6
•I-4 高病原H5N1禽流感的流行病學及臨床症狀 7

§II. 微脂粒簡介 9
•II-1 微脂粒的結構與組成 10
•II-2 微脂粒的分類 13
•II-3 微脂粒基本的製備方法 14
•II-4 微脂粒的性質 14
•II-5 微脂粒的穩定度 15
•II-6 微脂粒與佐劑的關係 16
＊II-6-1 佐劑 16
＊II-6-2 理想之黏膜佐劑免疫反應特性 18
•II-7 微脂粒之佐劑機制 19

§III . 水膠 20

•III-1 褐藻膠之結構 21
•III-2 褐藻膠之凝膠性質 22
•III-3 影響褐藻膠體強度之因子 24
＊III-3-1 褐藻酸鈉種類 24
＊III-3-2 褐藻酸鈉濃度 24
＊III-3-3 氯化鈣濃度 24
＊III-3-4 浸泡於鈣離子中之時間 25
＊III-3-5 其他離子 25
＊III-3-6 ｐH 值 26

§IV . 銀耳多醣的介紹 27

•IV-1 銀耳多醣的組成分 27
•IV-2 銀耳多醣的生理活性 29

§V. 免疫系統與黏膜免疫之簡介 30
•V-1 銀耳多醣的組成分 31
•V-2 銀耳多醣的生理活性 32

第三章 研究動機 34

第四章 實驗設計流程 35

第五章 實驗設備與實驗方法 36

§5-1 實驗材料與藥品 36

§5-2 實驗儀器與設備 38
•5-2-1 粒徑與界面電位分析儀 38
•5-2-2 減壓濃縮機 38
•5-2-3 其他設備儀器 39

§5-3 實驗方法 40
•5-3-1 溶液配製 40
•5-3-2 疫苗製備 42
•5-3-3 粒徑分析 43
•5-3-4 界面電位測定 44
•5-3-5 體外溶離實驗 44
•5-4-6 TEER試驗 45
•5-4-7 動物實驗 45
•5-4-8 抗體分析(血清、鼻腔沖洗液) 48
•5-4-9 血球凝集試驗HA test 49
•5-4-10 血球抑制凝集試驗HI test 49

第六章 結果與討論 50

•6-1 粒徑與介面電位分析 50
•6-2 體外溶離實驗 51
•6-3 Transepithelial Electrical Resistance, TEER試驗 54
•6-4 動物實驗分析 55
•6-4-1 血清中抗體分析 56
•6-4-2 小腸沖洗液之抗體sIgA分析 61
•6-4-3 血球凝集抑制實驗 62

第七章 結論 64

第八章 未來展望 65

第九章 參考文獻 66

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