臺灣博碩士論文加值系統

已知Glutathione S-transferase（GST）為一良好抗血吸蟲病的疫苗候選者。為瞭解宿主在感染日本血吸蟲後體內免疫反應和TH1及TH2細胞之相互關係，本實驗室曾以RT-PCR（Reverse Transcriptase Polymerase Chain Reaction）及ELISA方法分析宿主體內T細胞所產生之細胞激素之種類、活性及動力學反應。TH1及TH2細胞雖產生不同之cytokine，然無法直接得知體內TH1及TH2細胞的產生情形，主要是TH1和TH2細胞表面無精確可區分的表面抗原。本論文藉由本校微免所謝世良老師提供的TH1/TH2表面標記基因轉殖小鼠，研究台灣株日本血吸蟲26KDa GST疫苗抗原在宿主體內的免疫調節機轉及其免疫病理反應。研究結果顯示，小鼠經GST免疫後感染日本血吸蟲，其體內產生的TH1細胞在感染初期（第4週）有明顯上升；而在感染急性期（第7週），TH2細胞有大量的表現。B細胞在感染急性期（第12週）有大量的表現；NK細胞在感染初期（第4週）與急性期（第9週）皆有大量的表現。實驗結果證明應用基因轉殖小鼠模式可直接分析宿主經免疫注射後其體內TH1/TH2細胞及相關effector cells之產生及免疫調節現象。

Glutathione S-transferase (GST) has been recognized as one of the schistosme vaccine candidate. In order to elucidate the immune response against Schistosome japonicum in infected hosts, relationships between TH1 and TH2 cells, our laboratory used RT-PCR and ELISA to analyze the type, activities, and kinetics of splenic TH1 and TH2 cytokine responses. Although TH1 and TH2 cells produce different kind of cytokine, their specific surface antigens have not yet been identified for distinguishing between the two cell types. Recently, we have used a transgenic mice model which can express the TH1 and TH2 specific surface markers established by Dr. Shie-Liang Hsieh to investigate the protective immune mechanisms and immunopathogenesis brought about by the 26KDa GST vaccine antigen from a Taiwanese strain of S. japonicum. The result showed that TH1 cells increased significantly in the early stage (4 wk). However, the TH2 cells reached maximum in the acute stage (7 wk). B cells reached the peak at wk 12; NK cells increased markedly at wk 4 and reached maximum at wk 9. This study indicated that the application of transgenic mice model can directly measure TH1/TH2 and other effector cell production conducted by flow cytometric assay and the protective immune mechanisms of 26KDa GST were further clarificed.

中文摘要…………………………………………………………… 1
英文摘要…………………………………………………………… 2
第一章 緒論………………………………………………………. 3
第二章 材料與方法……………………………………………… 12
TH1與TH2基因轉殖小鼠之建立…………………… 12
TH1與TH2基因轉殖小鼠之篩檢…………………… 12
日本血吸蟲生活史之維持……………………………. 13
重組麩胺基硫S-轉移（rGST）蛋白的純化……… 14
成蟲可溶性蛋白之製備……………………………….. 15
抗血清之製備………………………………………….. 15
連免疫吸附分析（Enzyme-Linked Immunosorbent…
Assay; 簡稱ELISA）…………………………………. 15
SDS-PAGE與西方墨點法…………………………….. 16
26 KDa GST免疫保護機轉實驗……………………… 17
淋巴細胞的取得與細胞表面標記的測定……………. 17
肝臟之冷凍切片及組織化學染色……………………. 18
第三章 結果……………………………………………………... 20
TH1/TH2基因轉殖小鼠之確定……………………… 20
成蟲可溶性蛋白之製備………………………………. 20
rGST 26 KDa之表現與純化…………………………. 20
免疫小鼠血清IgG抗體力價之分析…………………. 21
多源抗體對rGST 26 KDa蛋白之專一性…………… 21
多源抗體對rGST 26 KDa蛋白之專一性…………… 21
CD4+細胞之表現情形……………………………….. 22
CD8+細胞之表現情形……………………………….. 22
TH1細胞之表現情形………………………………… 23
TH2細胞之表現情形………………………………… 24
B細胞之表現情形……………………………………. 24
NK細胞之表現情形………………………………….. 25
小鼠感染日本血吸蟲後之病理變化…………………. 25
第四章 討 論…………………………………………………….. 27
致謝………………………………………………………………… 33
參考文獻…………………………………………………………… 34
圖表及說明………………………………………………………… 42

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