臺灣博碩士論文加值系統

本論文以同步噴霧乾燥法(co-spray drying method)製作微膠囊口服疫苗，以豬肺炎黴漿菌作為核心物質，Eudragit L30 D55及Aquacoat ECD作為包覆材，將核心物質、包覆材及其他添加物事先混勻，用同步噴霧乾燥法方式製成微膠囊，經實驗室測定物理及化學特性後，再以動物試驗評估其免疫及保護效果。另外為了提升微膠囊疫苗的使用效率，以佐劑添加至微膠囊中，再進行動物試驗以評估其效果。
所製得微膠囊最適合的保存溫度為4℃，在此溫度下，配方T1E15儲藏時間為1個月而配方AS-HF為3個月。在不同pH值下溶離曲線觀察，配方T1E15在pH 5以上就會快速釋放，而配方AS-HF要在pH 6以上才會釋出蛋白質，但兩種配方都符合美國藥典所規定腸溶性藥物釋放模式。不同製程特性比較試驗下，以同步噴霧乾燥法(配方AS-HF)與溶液蒸發法(配方CAP)製程所得微膠囊來進行包覆率測定，發現噴霧乾燥法的包覆率(>93%)遠較溶液蒸發法包覆率(<35%)為高。在小鼠及豬隻試驗中，三種微膠囊口服疫苗的配方─T1E15、AS-HF、CAP同樣都可引起免疫反應，而經過M. hyopneumoniae攻毒後的保護試驗則顯示以配方CAP及配方AS-HF效果最好，其次為T1E15，表示豬黴漿菌微膠囊口服疫苗確實可有效預防豬黴漿菌肺炎發生。為避免口服疫苗產生免疫耐受性，以大腸桿菌忌熱性毒素加入微膠囊中，在小鼠試驗顯示確實可以在低劑量時誘發免疫反應，在高劑量時協助提高免疫耐受性的劑量，而在豬隻試驗中也表現出增強免疫反應的作用。
利用已經建立的微膠囊模式，將豬霍亂沙門氏菌製成微膠囊口服疫苗，與aroA變異株減毒活菌疫苗一同進行小鼠免疫及攻毒試驗，發現aroA變異株活菌疫苗效果最佳，雖然沒有顯著的體液性免疫反應，但保護率卻可達百分之百。反觀微膠囊口服疫苗組雖可成功誘發抗體，可是保護率卻只有60%，顯示尚有改善的餘地。 未來工作進行有二，一為將微膠囊應用於其他病原，製造出多價的微膠囊口服疫苗；二為開發更具有效率的微膠囊疫苗，使疫苗的保護效果更好。

In this study, M. hyopneumoniae was used as core materials and Eudragit L30 D55 or Aquacoat ECD was used as a coating material to produce microspheres by co-spray drying process. The characterization of microspheres was evaluated with physicochemical methods in vitro and immunogenicity of the entrapped antigen was determinate in vivo. Adjuvant was also added to microspheres vaccine for increasing the efficiency of vaccination.
In vitro study revealed that the optimum storage temperature was 4°C. The storage time for T1E15 is one month, while that for AS-HF is three months. Under different pH values, T1E15 releases protein rapidly at a pH of 5 or above and AS-HF releases protein at a pH of 6 or above. Both formulations are consistent with U.S.A. drug regulations. Observing the surfaces of the microspheres in acid and neutral environment under an electron microscope, the surfaces remain unchanged in acid environments but display polypore and start to become damaged in neutral environments. Comparing the efficiency of the encapsulation of microspheres by solvent evaporation and co-spray drying, the efficiency of encapsulation by the co-spray drying method exceeds than 93%, higher than by solvent evaporation. The formulations T1E15 and AS-HF successfully induced anti-M. hyopneumoniae antibody in an experiment involving mice. Moreover, in experiments involving pigs, three oral microspheres formulations (T1E15, AS-HF, and CAP) also induced a good immune response. Challenge testing demonstrates that the CAP and AS- HF formulation provides the best protection, followed by T1E15. Accordingly, M. hyopneumoniae oral vaccines are efficient to prevent mycoplasma pneumonia. The experiments faced various problems, including immunology suppression and oral tolerance. To resolve these problems, E. coli heat labile toxin (LT) was added to the microspheres. The data of mice showed that LT could induce immune responses in lower doses and help increase oral tolerances in higher dose. In a swine model, LT also increased immune reactions in experiments involving pigs.
Based on this success in applying the M. hyopneumoniae vaccine, we hope that this vaccine can be applied successfully to controlling Salmonella choleraesuis. The experiment result shows that oral live vaccine provides the best protection for reaching a protection rate of 100%, although no obvious increase in humoral immune response is noted. Although microspheres oral vaccine successfully induces the antibody titer, it only provides 60% protection. The vaccine thus is not perfect, and will continue to be researched and improved to achieve the best formulation.

第一章 文獻探討
第一節 呼吸道與消化道免疫的關係─泛黏膜免疫系統
第二節 微粒包覆的緣由
第三節 豬黴漿菌肺炎
第四節 口服疫苗
第五節 腸溶性包覆材
第六節 噴霧乾燥法
第七節 口服耐受性
第八節 增加黏膜免疫的佐劑
第二章 Mycoplasma hyopneumoniae之抗原特性及其定量
第一節 摘要
第二節 緒言
第三節 材料與方法
第四節 結果與討論
第五節 結論
第三章 腸溶性Mycoplasma hyopneumoniae微膠囊口服疫苗之製備─包覆材與包覆方式比較
第一節 摘要
第二節 緒言
第三節 材料與方法
第四節 結果與討論
第五節 結論
第四章 Mycoplasma hyopneumoniae微膠囊口服疫苗之免疫保護試驗
第一節 摘要
第二節 緒言
第三節 材料與方法
第四節 結果與討論
第五節 結論
第五章 含佐劑微膠囊口服疫苗之效力試驗
第一節 摘要
第二節 緒言
第三節 材料與方法
第四節 結果與討論
第五節 結論
第六章 含Salmonella choleraesuis微膠囊口服疫苗之效力試驗
第一節 摘要
第二節 緒言
第三節 材料與方法
第四節 結果與討論
第五節 結論
第七章 總結與展望
第一節 總結
第二節 未來工作之展望
參考文獻
附錄

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