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研究生:鄭柏青
研究生(外文):Po-Ching Cheng
論文名稱:日本血吸蟲基因體學與免疫學研究
論文名稱(外文):Genomic and Immunological Studies of Schistosoma japonicum
指導教授:蔡洪又欽李金木李金木引用關係
指導教授(外文):John Chin Tsaihong,Kin Mu Lee
學位類別:博士
校院名稱:國立陽明大學
系所名稱:熱帶醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:168
中文關鍵詞:日本血吸蟲cDNA微陣列巨噬細胞基因體學血清診斷重組26kDaGST樟芝免疫調節T1/T2雙基因轉殖小鼠
外文關鍵詞:Schistosoma japonicumcDNA microarraymacrophagegenomicserodiagnosisreSjc26GSTAntrodia camphorataimmunomodulationT1/T2 doubly transgenic mice
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血吸蟲病至今仍是全球嚴重的公共衛生問題。常見的寄生於人體之三種血吸蟲,以日本血吸蟲產卵量最多,宿主專一性最差,因此所造成的危害也最嚴重。全世界已投入大量的資源於各種防治及研究計劃中,然而此病依然在流行區域持續地傳播著。雖然歷經許多學者專家長期的研究,血吸蟲病許多特性卻仍然不清楚且令人困惑。在本研究中,我們企圖透過多方面的探討,包括基因體學,免疫學及實際的應用科學對血吸蟲提出深入的解釋與新的觀點。本論文主要包括三個部分,茲敘述於下。
第一部分係以小鼠為模式探討巨噬細胞在血吸蟲感染過程中所扮演的角色。我們使用cDNA微陣列偵測小鼠感染血吸蟲不同階段的巨噬細胞整體性基因轉錄表現相關之變化,以瞭解寄生蟲與宿主之間的交互作用機制。同時利用流式細胞儀(Flow cytometry)探討小鼠感染日本血吸蟲後,巨噬細胞族群比例與表現標記變化,以及應用T1/T2雙基因轉殖小鼠觀察免疫整體性分化表現情形,進一步探討抗原呈現細胞與淋巴細胞的之共同作用關係。利用Hierarchical clustering分析證實巨噬細胞的基因表型於感染後4.5週到8週間,由Type 1型反應相關轉型變化為Type 2型免疫反應,且蟲卵出現之後的3個時間點基因規模與型式都相當類似。結果也顯示與TLR、IFN、MHC以及Tnfrsf等相關基因表現也整體地在4.5週到8週這時間點後受到抑制。推測這些免疫調節相關的基因在血吸蟲蟲卵及其引發的肉芽腫病變反應中受到調控下降。透過這些作用因子與受體間相互消長,巨噬細胞調節反應的強度與規模並控制病變狀況。特別的是,Alternatively activated macrophages(AAM���w指標基因YM1及YM2在蟲卵出現後的感染過程中逐漸增加表現,顯示AAM�眥捋P降低肝臟肉芽腫發炎及長期減緩猛烈細胞毒殺等會造成組織傷害的Th1免疫反應有關。基因表現型態分析也確立巨噬細胞在轉型前調控發炎或細胞毒殺相關基因,而轉型後則調控趨化、黏附與組織修復等相關功能基因。
組織病理分析也證實第7週急性期後,肝臟組織迅速出現免疫細胞浸潤與肉芽腫形成等蟲卵造成的病變,並隨著時間嚴重。另外流式細胞儀分析試驗顯示巨噬細胞除了在第3及5週表現較為下降外,其他時間點均明顯偏高,而其表面標記MHC class II (IA/IE)與CD8α+也在蟲卵出現前後發生戲劇性的改變消長。而基因轉殖小鼠實驗也證實CD4+ T與B 細胞在血吸蟲感染過程發生比例變化及Th1/Th2分化。Type1細胞僅在3-5週上升,但是在蟲卵出現後Th2及Be2細胞即明顯增加。結果顯示可能日本血吸蟲感染過程中,抗原影響巨噬細胞的替代活化表型轉換,藉此調控了後續淋巴細胞的分化與免疫的走向。以上所述,闡釋了巨噬細胞的分化與血吸蟲不同感染階段的關係,更提供了感染宿主巨噬細胞參與調控免疫網絡的可能機制。此研究的結果將有助於未來日本血吸蟲免疫學與基因體學更進一步的相關研究。
第二部分,為了建立有效的牛隻感染日本血吸蟲症診斷方法,我們clone Sjc26GST(26kDa Glutathione S-transferase of Schistosoma japonicum from Chinese strain)基因並以E. coli表現製備出具His-tag的GST重組蛋白。所純化的重組Sjc26GST蛋白利用酵素連結免疫吸附分析(ELISA)與免疫墨點法在水牛群中測試是否感染日本血吸蟲。測試結果顯示疫區中血吸蟲蟲卵陽性的牛隻血清,全部呈現陽性反應;在疫區陰性組別中亦有30.3%出現陽性;然而在非疫區的牛隻則完全出現陰性反應。疫區蟲卵陽性組及陰性組別牛隻的專一性抗體之平均O.D.值分別為非疫區牛隻的3.36倍及1.69倍。牛隻測試陽性之血清以西方墨點法分析可辨認出一明顯的27 kDa蛋白,即為所純化的reSjc26GST。本研究結果顯示以E. coli表現的重組GST蛋白因製備方便,且靈敏度與專一性高,具有做為日本血吸蟲診斷試劑的價值。
第三部分,由於血吸蟲的感染程度與宿主本身之免疫反應能力成絕對的關係,因此我們利用活體實驗動物模式,以血吸蟲獨特的慢性感染特質來評估中草藥的免疫調節功效。在研究中以樟芝多醣體分別餵食C57BL/6j、BALB/c及T1/T2雙基因轉殖小鼠後,藉由流式細胞儀及RT-PCR技術來探討多醣體對小鼠所產生之細胞族群中表面標記及細胞激素的影響變化;最後應用曼氏血吸蟲感染小鼠模式,進行總體保護力提升之評估。在RT-PCR之細胞激素分析中,餵食樟芝多醣體的C57BL/6j或BALB/c小鼠其體內IFN-�蛂BIL-2以及TNF-�� mRNA之levels都呈現明顯增加的趨勢。而以流式細胞儀在T1/T2雙基因轉殖小鼠餵食樟芝多醣體的實驗中,CD4 T細胞及B細胞均有明顯增強的效果。更特殊的是,觀察這些細胞的Type1/Type2標記表現,其中Th1與Be1細胞有明顯增加的情形;顯示樟芝對於Type1系列之淋巴細胞有明顯提升的效果。而BALB/c小鼠餵食樟芝多醣六週後,脾臟DC與巨噬細胞的數量以及DC表面標記CD8α+及MHC class II I-A/I-E都有增加。在樟芝多醣體對小鼠保護力的評估中,餵食二週的BALB/c小鼠所得之血吸蟲成蟲數與對照組比較,即有明顯下降的趨勢;而在餵食四週後,在C56BL/6j及BALB/c小鼠中均呈現明顯降低之趨勢。此結果顯示經服用樟芝多醣體後,能透過DC細胞作用增強小鼠體內免疫系統Th1機制的細胞數量及活性,進而提升其所增強的免疫療效能在活體內發揮明顯的功能,以預防抑制曼氏血吸蟲此寄生蟲病源的感染。
總而言之,本論文不但以基因體學與免疫學基礎探討血吸蟲與宿主的關係,特別是巨噬細胞參與調節的機制。此外,更發展一新的診斷技術應用於家畜的血吸蟲感染症,以及一新應用於中草藥的血吸蟲感染免疫調節功效評估模式。我們相信藉由這些廣泛的研究,將爲未來血吸蟲研究的方法與方向與提供新里程碑。
Schistosomiasis is continuing a serious problem in public health around the world. This parasitic disease is mainly caused by the release of a large number of eggs by adult worms. Schistosoma japonicum infection induces severe pathological lesions because the large produce the most counts of eggs. Although a large amount of resources have been invested in various control and research programs, transmission remains in most of the endemic areas. Some characteristics of the worm are still unclear and have long perplexed investigators intent on controlling this parasitic disease. In the present thesis, we have conducted extensive studies and discussions on multifaceted aspects of genomics, immunology, and actual applications of Schistosoma. The entire thesis includes 3 sections as provided below.

The first and main section reveals that macrophages play crucial roles in immune responses as they can initiate, modulate, and also serve as final effector cells during course of schistosomal infections. For better understanding the innate immune responses during host-parasite interactions, the global gene expression profiles of macrophages from the spleen of mice at different infection stages were compared by microarray-based analysis. Concurrently, we not only used flow cytometric analysis to investigate the immunocompetent changes in the macrophages, but also evaluated the population-differentiated proportion of the immune phenotype in a unique T1/T2 double transgenic mouse model. Hierarchical clustering analysis demonstrated that a significant switch in gene transformation associated with a type-1 response and linked with a type-2 cytokine phenotype occurs between 4.5 and 8 weeks post-infection. Moreover, the gene profiles at 3 later time-points following egg challenge were similar in complexity and magnitude. These data also showed that there are mostly inhibtion in gene expression related TLRs, IFN, MHC and TNFrsf at the switch between 4.5 and 8 weeks post-infection, and it is suggested that these immunomodulatory genes may be downregulated in resistance against S. japonicum eggs and granuloma pathology. In particular, the gradual upregulation of genes encoding YM1 and YM2 in infected mice indicated the role of alternatively activated macrophages (AAM�珺) in immune responses after schistosoma egg production. The induction of AAM�珺 in the spleen could be important for dampening the level of inflammation in hepatic granulomas and contributing to a long-term decrease in violent cytotoxicity and tissue-damaging immune responses that are associated with type-1 immunity. The genes expressions involved in repair/remodeling during liver fibrosis were also observed after eggs production. Histological analysis demonstrated that cellular infiltration and granuloma formation were initiated quickly after 7-week post-infection as a result of S. japonicum eggs deposited into the liver, and the pathology of disease gradually progressed with time. Flow cytometric analysis showed that macrophages from mice infected with S. japonicum were decreased at 3-5 weeks, and the number re-increased during eggs production at 7 weeks p.i. The surface markers MHC class II (IA/IE) and CD8α+ of the macrophages also exhibited a dramatic change before and after the egg-production time points. The transgenic mice experiments further demonstrated the proportion of CD4+ T cells and B cells shifted and differentiated into type 1 and type 2 regulatory subpopulations over the course of infection. The percentage of CD4+ Th2 cells was observed to be much higher than that of Th1 cells, especially after stimulation with S. japonicum eggs, whereas type 1 cells increased only at 3 and 5 weeks post-infection. The differentiation of B cells showed a similar trend. These results suggested that the infection of mice with S. japonicum resulted in a final Th2-skewed immune response that was associated with the phenotypical changes in the macrophages and the influence of macrophages that were alternatively activated by cytokine cross-modulation. All of these analyses elucidated a correlation between macrophage differentiation in systemic immune responses and different stages of schistosoma infection, thus proving the existence of variation in innate and adaptive immune mechanisms at the different infection stages. Understanding these immune mechanisms related to parasite resistance, pathology, and growth with regard to the disease will be helpful in further studies on S. japonicum.

In the second part, we applied recombinant Sjc26GST protein for serodiagnosis of S. japonicum infection in water buffalo. The gene encoding Sjc26GST was cloned and expressed in Escherichia coli as a fusion protein with a His-tag. The purified reSjc26GST was used as an antigen for an enzyme-linked immunosorbent assay (ELISA) and for immunoblotting to detect S. japonicum antibodies in water buffaloes. Our results showed that mean the O.D. values of specific serum IgG antibodies from egg-positive buffaloes were 3.37-fold higher than that found in egg-negative buffaloes from non-endemic areas. The data also showed that the O.D. value of the endemic egg-negative group reached as high as 1.69 times that found in the non-endemic areas. The positivity rate of egg-positive buffaloes was 100%, but was 30.3% in the endemic egg-negative group. Infected bovine antisera also recognized reSjc26GST, a 27 kDa protein as determined by Western blot. These results suggest that the recombinant GST expressed in E. coli would be an effective diagnostic reagent for the detection of antibodies against S. japonicum in buffaloes. Due to its easy production, excellent sensitivity, and high specificity, the reSjc26GST protein described in this study can be considered as a candidate protein for immunological diagnosis of bovine schistosomiasis.

In the third section, we attempted to establish an in vivo schistosomal mouse infection model to study the immunomodulatory roles of Chinese herbal medicine. The unique chronic characteristic of this parasite are suited to elucidate gentle curative efficacy of Chinese herbs. In this study, we tested the in vivo effects of polysaccharides derived from A. camphorata (AC-PS) on the immune function by detection of cytokine expression, and evaluation of the immune phenotype in inbred mice or the T1/T2 doubly transgenic mouse model. The protective effect of AC-PS in mice was finally tested by infection with S. mansoni. The induction of large amounts of IFN-γ, IL-2, and TNF-α mRNA were detected after 2 and 4 weeks of oral AC-PS administration in BALB/c and C57BL/6j mice. After 6 weeks of oral AC-PS administration to BALB/c mice, the number of splenic dendritic cells, macrophages as well as the surface expression of CD8α+ and MHC class II I-A/I-E on dendritic cells increased. The CD4+/CD8+ T cells ratio and number of B cells among the splenocytes were also augmented. In transgenic mice, 3- to 6-weeks of oral AC-PS administration increased the proportion of CD4+ T cells and B cells in the spleen. More specifically, there was an increase in Th1 CD4+ T cells and Be1 cells among the spleen cells as observed by detection of type1/type2 marker molecules. Using a disease model for parasitic infection, we found that AC-PS treatment inhibited S. mansoni infection in BALB/c and C57BL/6j mice. AC-PS appears to influence the development of Th1 responses in the immune system and has the potential for preventing S. mansoni infection.

Taken together, this thesis had led to a profound understanding of schistosome-host interactions on a genomic and immunological basis, especially with regard to innate immunity of macrophages. This thesis also provides a new diagnostic method for domestic schistosomiasis and a unique in vivo schistosomal infection model for evaluating the curative efficacy of drugs or health food. We believe that these extensive studies result in improvements and provide direction of further schistosome investigations.
中文摘要………………………………………………………………………....1
英文摘要………………………………………………………………………....4
第一章 總緒論………………………………………………………………….7
1-1 前言……………………………………………………………….......8
1-2 研究目的…………………………………………………………….11
1-3 參考文獻…………………………………………………………….12
第二章 應用微陣列方法分析血吸蟲感染不同期別宿主巨噬細胞免疫調控基因反應之研究……………………………………………………...14
2-1 緒言………………………………………………………………….15
2-2 材料與方法………………………………………………………….19
2-3 結果………………………………………………………………….24
2-4 討論………………………………………………………………….38
2-5 參考文獻…………………………………………………………….46
2-6 圖表………………………………………………………………….60
第三章 應用重組Sjc26GST開發牛隻感染日本血吸蟲之血清診斷研究………………………………………………………………….....102
3-1 緒言…………………………………………………………...…...103
3-2 材料與方法………………………………………………………..105
3-3 結果……………………………………………………………......108
3-4 討論………………………………………………………………..110
3-5 參考文獻…………………………………………………………..112
3-6 圖表………………………………………………………………..115
第四章 以血吸蟲感染評估樟芝多醣體對小鼠免疫調控之功效………….119
4-1 緒言………………………………………………………………..120
4-2 材料與方法………………………………………………………..123
4-3 結果………………………………………………………………..128
4-4 討論………………………………………………………………..131
4-5 參考文獻…………………………………………………………..134
4-6 圖表………………………………………………………………..139
第五章 總結論……………………………………………………………….147
5-1 結論…………………………………………………………….....148
期刊附錄………………………………………………………………………150
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