摘要
幾丁聚醣(Chitosan)，由幾丁質(Chitin)俗稱的甲殼素，經過高濃度鹼液與高溫去乙醯化作用，得到的一種天然、無毒的高分子聚合物。可從大自然中取得，且能利用廢棄之蝦蟹殼之廢棄物來加工製成幾丁聚醣(Chitosan)，不影響環境生態，易被生物分解其產生的低聚醣也能夠被代謝，不會產生毒性危害生物體，近年來被廣泛使用於生物醫學，作為生物材料。其他研究顯示幾丁聚醣作為佐劑與輔助配方，能夠保護抗原並增加免疫刺激活性，如：刺激白血球釋出細胞激素、增加巨噬細胞的聚集和激活、增加抗感染和細胞毒性。用來作為生物吸附劑當作疫苗的佐劑成份，具有吸附抗原的特性能夠作為抗原的載體，也能夠提升免疫效果。幾丁聚醣為成本低廉可利用之自然資源，評估未來取代昂貴的傳統佐劑作為新疫苗佐劑的可行性。
因此，本研究中利用幾丁聚醣吸附抗原與增加免疫刺激活性的特性，使用奈米化的幾丁聚醣顆粒吸附抗原腸病毒EV71，經過純化、透析，測試良好的調劑條件後將其製備為抗腸病毒EV71疫苗，施打於小鼠進行動物實驗，觀察小鼠免疫後的反應，測試包覆腸病毒之奈米化幾丁聚醣顆粒能否誘發較好的抗腸病毒EV71免疫反應。
關鍵詞 ： 幾丁質、腸病毒、幾丁聚醣、佐劑

Abstract
Chitin, a natural and non-toxic polymer, can be obtained from shrimp and crab shell waste. After deacetylation with high concentrations of lye and high temperature, chitin can be processed into chitosan. Chitosan does not affect the environment and ecology. With biodegradable and non-toxic properties, chitosan has been widely used in biomedical as a biological material in recent years. Many studies have shown that chitosan can be as adjuvant and adjuvant formula to protect the antigen and increase the immune stimulus activity, such as stimulation of white blood cells to release cytokines, the increase of macrophage aggregation and activation, the increase of anti-infection and cytotoxicity. The chitosan adjuvant component being a biosorbent has the ability to adsorb the antigen as a vaccine vector and also enhance the immune effect. Chitosan, therefore, is a viable alternative to the use of natural resources to assess the feasibility of replacing traditional expensive adjuvants as new vaccine adjuvants in the future.
In this study, we used nano-chitosan particles to adsorb enterovirus EV71antigen. The activity of immunostimulatory activity of nano-chitosan particles with EV71 antigen was examined by animal experiments and the response of mice after immunization was observed.
Key word : Chitin、Enterovirus、Chitosan、Adjuvant

目錄
論文審定書 i
致謝 ii
摘要 iii
Abstract iv
目錄 v
圖次 vii
表次 viii
第一章 緒論 1
1-1 幾丁質與幾丁聚醣的介紹 2
1-2 幾丁質與幾丁聚醣的結構 2
1-3 幾丁聚醣的應用 4
1-3-1 農業應用 4
1-3-2 食品加工 4
1-3-3 化工應用 5
1-3-4 醫學應用 5
1-4 腸病毒71型的簡介 8
第二章 材料與方法 11
2-1 實驗藥品 11
2-2 儀器設備 12
2-3 細胞繼代 13
2-4 細胞保存 13
2-5 細胞解凍 14
2-6 腸病毒71型培養及純化 14
2-7 病毒力價試驗 (TCID50) 14
2-8 病毒顆粒的透析 15
2-9 幾丁聚醣奈米顆粒的吸附 15
2-10 SDS-PAGE 16
2-11 西方點墨法(Western blotting) 18
2-12 動物實驗 20
2-13 血清中和試驗 22
2-14 統計分析 22
第三章 結果與討論 23
3-1 腸病毒目標蛋白確認 23
3-2 顆粒製備最佳條件測試 23
3-2-1 顆粒製備-不同pH值對顆粒形成與病毒包覆的影響 23
3-2-2 顆粒製備-不同TPP濃度對顆粒形成與病毒包覆的影響 24
3-3 顆粒吸附病毒蛋白後SDS PAGE分析結果 24
3-3-1 分析不同濃度TPP顆粒之病毒包覆效果 25
3-4 動物實驗-小鼠血清中和抗體 25
第四章 結論與建議 27
第五章 參考文獻 29

衛生福利部疾病管制署，腸病毒專區，疫情消息

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