豬肺炎黴漿菌(Mycoplasma hypneumoniae, Mhp)為造成豬地方性肺炎(Enzootic pneumonia, EP)之主要病原，會引發高發病率但低死亡率之慢性呼吸道疾病。為防範此病，疫苗施打為目前主要策略。疫苗所誘發的特異性抗體除了可阻斷病原感染，亦可作為調理素促使巨噬細胞之吞噬作用，因此抗原誘發之免疫保護效果對疫苗開發具重要性。本研究主要目的為篩選出良好之豬肺炎黴漿菌抗原使之誘發有效抗體，以提高疫苗免疫保護效力，並且以小鼠模式作為取代動物之可行性測試。試驗首先建立豬肺泡巨噬細胞(PAM)吞噬不活化豬肺炎黴漿菌之方法學，以顯微鏡確認螢光標定之豬肺炎黴漿菌可被PAM吞噬後，以不同濃度之Mhp與吞噬時間進行條件最適化。發現當Mhp濃度為200 ng/ml而吞噬時間為30分鐘時，PAM的吞噬量最為合適。而後對免疫豬肺炎黴漿菌死菌疫苗前後之血清進行吞噬量的評估，發現免疫後之吞噬量顯著增加。並在單抗原次單位疫苗中發現，以Mhp-1與Mhp-6能明顯的增加PAM的吞噬能力。試驗接著將各單抗原免疫後之血清以不同排列組合方式進行混合後再進行吞噬試驗，結果顯示，吞噬量最佳之前十個血清組合皆包含抗原Mhp-1，且發現混合的血清種類越多，PAM的吞噬能力也隨之增加。後續試驗將六種抗原混合製成豬肺炎黴漿菌次單位疫苗，並且同步測試純化與非純化之抗原是否有差異。結果發現，免疫次單位及純化次單位之疫苗具相同效果，皆可顯著增加PAM的吞噬能力。試驗最後建立小鼠巨噬細胞( RAW 264.7 )吞噬方法學，以此方法平行比對上述試驗。發現不論是在免疫血清試驗或是比較各血清組合，結果皆證實，RAW 264.7與PAM吞噬試驗有相同之趨勢。綜合上述，可利用巨噬細胞吞噬能力評估疫苗免疫保護效力，以此方法篩選出有效抗原，並可以小鼠作為取代評估之動物模式。

Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) is the etiological agent of enzootic pneumonia, a chronic respiratory disease characterized by high morbidity and low mortality. Currently, vaccination is the primary strategy to prevent this disease. Vaccine-induced specific antibodies in addition to blocking the pathogen, but also as a opsonin to promote macrophage phagocytosis. The protection differences conferred by different antigens have important implications with vaccine potential. The goals of this study was to select suitable antigen to improve the immune protective efficacy of Mhp vaccine. We demonstrated the engulment of FITC-labeled inactivated Mhp by porcine alveolar macrophages (PAM), and then revealed that the phagocytosis of PAM was most appropriate when treatment with 200 ng/ml Mhp for 30 minutes. Interestingly, we found that the phagocytosis of PAM was increased significantly after vaccination. In single-antigen subunit-based vaccines tests, Mhp-1 and Mhp-6 were found to significantly increase the phagocytosis of PAM. When testing different permutations and combinations of post-vaccination serum samples for the efficiency of PAM phagocytosis, we observed that the top ten most efficient samples all contain antigen Mhp-1, and also revealed that the more mixed serum types yields the better ability of phagocytosis. Subsequently, six Mhp antigens were mixed as subunit vaccine and were subjected for comparing the difference between purified and non-purified antigens, and we found that there is no difference in the stimulation of PAM-mediated phagocytosis. Lastly, we examined mouse macrophages (RAW 264.7) as the model system for phagocytosis of inactivated Mhp in comparison with PAM-mediated phagocytosis, and demonstrated the similar trends between the phagocytosis of RAW 264.7 and PAM. In conclusion, we validated macrophage-mediated phagocytosis can select suitable antigen to improve the immune protective efficacy of Mhp vaccine, and verified the feasibility of mouse model as substituted testing.

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中文摘要…………………………………………………………………….……. …... i
英文摘要……………………………………………………………………………. ii
目次……………………………………………………………………………………. iii
圖次……………………………………………………………………………………. vi
表次………………………………………………………………………………......... vii
第一章 前言……………………………………………….………………………....... 1
第二章 文獻探討………………………………………….………………………....... 2
第一節 豬肺炎黴漿菌…………………………………………………………… 2
1.1 簡介與特性……………………………………....................................... 2
1.2 臨床症狀…………………………………………….............................. 2
1.3 致病機制……………………………………………….......................... 2
1.4 預防控制…………………………………………………...................... 3
第二節 呼吸道免疫系統…………………………………………………………
…………3
2.1 簡介……………….………………........................................................... 3
2.2 特異性免疫機制…………………………………………...................... 3
2.3 巨噬細胞………………………………………....................................... 4
第三節 吞噬作用……………………………..…………………………………. 4
3.1 簡介……………………………………………...................................... 4
3.2 吞噬體形成………………….………………………………………… 5
3.3 吞噬體成熟……………………………………………………………. 5
3.4 吞噬溶酶體……………………………………………………………. 6
3.4.1 酸化……………………………………………………………... 6
3.4.2 活性氧物質……………………………………………………... 6
3.5 吞噬評估方式………………………………………………………... 6
第四節 疫苗與吞噬作用之相關性………………………………………...……. 7
4.1 前人研究……………………….……………………............................. 7
4.2 小鼠模式……..………………………………………………………... 7
第三章 材料與方法………………………………………….………………………... 8
第一節 巨噬細胞製備與培養…………………………………………………… 8
1.1豬肺泡巨噬細胞…………………………………………….…………... 8

1.1.1 PAM灌洗收集與保存…………………….……………………... 8
1.1.2 PAM培養….…………………………….………………………. 8
1.2 小鼠巨噬細胞…………………………………………………………... 9
1.2.1 RAW 264.7解凍與培養.………..…………..................................... 9
1.2.2 RAW 264.7繼代……….……………………………...…….......... 9
第二節 豬肺炎黴漿菌菌株螢光標示………..……………………........................ 9
2.1 豬肺炎黴漿菌培養與不活化…….…………………………………….. 9
2.1.1 不活化劑配製…………….……………………………………... 9
2.1.2 菌液培養與不活化測試…..…….………………………………. 10
2.2 菌株螢光標示..…………………………………………………………. 10
2.2.1 螢光標示液配製………………………………………………… 10
2.2.2 螢光標示…………………….…………………………………... 10
2.3. 蛋白質濃度測定..……………………………………………………… 10
第三節 巨噬細胞吞噬不活化豬肺炎黴漿菌方法學建立………………………. 11
3.1 螢光顯微鏡觀察……………..…………………………………………. 11
3.1.1 豬肺炎黴漿菌與細胞感作……………………………………… 11
3.1.2 細胞染色…………………….……………...…………………… 11
3.2 不同濃度豬肺炎黴漿菌對吞噬之影響..………….……………………. 11
3.3 不同吞噬時間對吞噬之影響………………………..…………………. 12
3.4 以流式細胞儀偵測細胞所含之螢光強度…………………...…….…… 12
第四節 動物試驗………………………………………………………………… 12
4.1 豬隻動物試驗……………..……………………………………………. 12
4.1.1 豬隻來源及申請…………………….…………………………... 12
4.1.2 豬隻試驗設計………………….…………………………...…… 12
4.2 小鼠動物試驗……………..……………………………………………. 13
4.2.1 小鼠來源及申請…………………….…………………………... 13
4.2.2 小鼠試驗設計…………..……………............................................ 13
4.3 吞噬試驗………………....…………………............................................. 13
第五節 統計分析………………………………………………………………… 13
第四章 結果…………………………………………………………………………... 14
第一節 製備螢光標示之不活化豬肺炎黴漿菌………………………...………. 14
1.1 豬肺炎黴漿菌不活化條件之探討………...……………………....…… 14

1.2 豬肺炎黴漿菌定量..……………………………………………………. 14
第二節 豬隻巨噬細胞吞噬豬肺炎黴漿菌系統………………………………… 14
2.1 巨噬細胞吞噬不活化豬肺炎黴漿菌方法學建立……………………… 14
2.1.1 巨噬細胞吞噬豬肺炎黴漿菌之螢光染色結果……...………….. 14
2.1.2 不同濃度之豬肺炎黴漿菌對PAM吞噬之影響………...……… 15
2.1.3 不同吞噬時間對PAM吞噬之影響…………...………………... 15
2.2. 豬隻動物試驗………..……….................................... 15
2.2.1 豬黴漿菌各單抗原對豬肺泡巨噬細胞吞噬之影響...………….. 15
2.2.2 不同抗原免疫後之血清組合….………….……………………... 16
2.2.3 豬黴漿菌疫苗對豬肺泡巨噬細胞吞噬之影響…………………. 16
第三節 小鼠巨噬細胞吞噬豬肺炎黴漿菌系統………………………………… 16
3.1 小鼠巨噬細胞吞噬不活化豬肺炎黴漿菌方法學建立............................. 16
3.1.1 巨噬細胞吞噬豬肺炎黴漿菌之螢光染色結果…………………. 16
3.1.2 不同濃度豬肺炎黴漿菌對RAW 264.7吞噬之影響…………… 17
3.1.3 不同吞噬時間對RAW 264.7吞噬之影響…………… 17
3.2 小鼠動物試驗………..……..……………………..................................... 17
3.2.1 豬黴漿菌各單抗原對小鼠巨噬細胞吞噬之影響………………. 17
3.2.2 不同抗原免疫後之血清組合….………………………………... 18
3.2.3 豬黴漿菌疫苗對小鼠巨噬細胞吞噬之影響……………………. 17
第五章 討論………………………………………………….………..……………… 19
參考文獻…………………………………………………….………………………… 40




 

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