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研究生:林裕翔
研究生(外文):Lin, Yu-Hsiang
論文名稱:探討Bacteroides fragilis菌對無菌鼠細胞免疫的影響
論文名稱(外文):Cellular Immunity Of Germ-free Mice Induced By Bacteroides fragilis
指導教授:王淑音王淑音引用關係黃彥智黃彥智引用關係
指導教授(外文):Wang, Shu-YinHuang, Yen-Te Evan
口試委員:黃彥智詹明修張春梵王淑音
口試委員(外文):Huang, Yen-Te EvanJan, Ming-ShiouChang, Chun-FanWang, Shu-Yin
口試日期:2012-06-21
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:64
中文關鍵詞:正常腸道微生物叢脆弱擬桿菌無菌小鼠單一已知菌動物
外文關鍵詞:normal microbiotaBacteroides fragilisGerm-free micemono-associated gnotobiotic mice
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正常腸道微生物叢 (normal microbiota)的存在,可以幫助宿主抵抗病原菌的侵襲及健全免疫力,甚至提供維生素等營養素。如果腸道菌叢發生變化或被破壞,就會給與伺機性病原菌(opportunistic pathogen) 造成宿主發生腸炎或感染的機會。本實驗為釐清腸道共生菌參與宿主的免疫調節作用,採用存在大部分哺乳動物腸道中之共生菌Bacteroides fragilis (B. fragilis),屬於絕對厭氧之格蘭氏陰性菌。以管餵方式定殖(colonization)到8至9週齡無菌小鼠(Germ-free mice, GF),於接菌四星期後犧牲並進行分析該菌對於無菌小鼠免疫系統之影響,對照組為不含B. fragilis (BF)之無菌小鼠。結果顯示:1.比較BF實驗組與GF對照組之體重並無顯著性差異,盲腸重量及結直腸長度則具有顯著性差異;2.由血球計數器計算週邊單核細胞 ( peripheral blood mononuclear cells, PBMC)結果得知,白血球數量在BF組與GF組小鼠並無明顯差異,中性球與單核球的比例則有顯著性增加;3.定殖四星期後,於盲腸與結腸之B. fragilis菌約為1010 cfu/g;4.以流式細胞儀分析脾臟、腸系膜淋巴結與培耶氏斑的T細胞與B細胞族群,結果發現投與B. fragilis四星期後可誘發腸系膜淋巴結T細胞與B細胞的比例有顯著性增加;5.分析結直腸細胞產生細胞素的能力,結果顯示,BF組小鼠抗炎症細胞素IL-10表現量顯著高於GF小鼠; 6. in vitro培養BF組小鼠脾臟細胞經LPS或Con A刺激釋放Th1 相關TNF-α與IFN-γ等細胞素的能力比GF小鼠高,相反的其釋放Th2相關細胞素如IL-4、IL-10則比GF小鼠低。综合上述結果顯示,B. fragilis具有調節無菌小鼠免疫的功能,並可藉由管餵方式建立B. fragilis單一已知菌動物(mono-associated gnotobiotic mice) 模式。
Normal flora are bacteria, fungi, and protozoa that live on or within the bodies of animals and plants. Usually, they do not cause disease in healthy individuals. Instead, they are commensalists or mutualists with regard to the host. That is, in addition to basically not harming the host, they can even do some good for prevention opportunistic pathogens. In order to investigate the immunological roles of symbiotic bacteria on intestine mucosal, germ-free 8- to 9-week old male C57BL/6JNarl mice , were housed under sterilized isolator and with free access to food and water. Mice were oral with 0.5 ml, 1108/per of Bacteroides fragilis (B. fragilis, BF) dissolved in saline, as well as oral with 0.5 ml saline as a control group. The animals were sacrificed at 1 month for physiology, gross and histopathology, immunohistochemistry, flow cytometry, cytokine, and PCR analysis. The results show no significant difference of body weight as well as WBC between both groups. However, the neutrophils and monocytes were induced in BF group. The lymphocytes were aggregated in lamina propria of BF’s intestine. In addition, the GALT hyperplasia was also noted. Under immunohistochemistry and flow cytometry, B cells in GALT, CD3 T cells in MLN were induced by B. fragilis. The production of IL-10 was also increased in colon. The splenocytes were stimulated by LPS or ConA in vitro. Interestingly, the production of TNF-α and IFN-γ (Th1) and reduction of IL-4 and IL-10 (Th2) were induced by B. fragilis. The results suggested that B. fragilis not only stimulated differention of local immunity but also modulated the systemic immune system. The mono-associated gnotobiotic mice model was first established in Taiwan.
摘要 1
Abstract 3
第一章、前言 5
第二章 文獻回顧 6
第一節 腸道共生菌與宿主免疫反應之相互關係 6
1. 共生菌Commensal bacteria (正常腸道菌叢Normal intestinal flora, NIF)與共生 Mutualism 6
2. 腸道相關淋巴組織 Gut-associated lymphoid tissue (GALT) 7
3. 輔助性T helper 1 (Th1)與T helper 2 (Th2)反應之調節 7
4. 共生菌與益生菌 8
第二節 無菌(germ-free, GF)與已知菌(gnotobiotic, GN)動物的特性 11
第三節 Bacteroides fragilis 與免疫系統之間的相互作用 13
1. Bacteroides fragilis (B. fragilis) 13
2. B. fragilis作為免疫調節抗原 13
3. B. fragilis誘發免疫系統成熟與Th1/Th2平衡 14
4. B. fragilis調節抗發炎反應 15
第三章 研究動機與目的 17
第四章 材料與方法 18
一、無菌動物之維持 18
二、Bacteroides fragilis 18
三、已知菌動物建立及分析 18
四、實驗方法 19
1. 菌量分析 19
2. 血球計數儀 (complete blood count) 19
3. 盲腸比率與形態學觀察 19
4. 免疫組織化學染色 (Immunohistochemistry) 19
5. 初代細胞培養 20
6. 流氏細胞儀分析(Flow Cytometry) 21
7. 酵素結合免疫分析法(Enzyme-linked Immunosorbent Assay;ELISA) 22
8. Real-time PCR 偵測細胞素和iNOS RNA表現量 22
9. 統計分析 24
第五章、結果 25
一、B. fragilis接菌後之菌量變化 25
二、盲腸比率與腸道形態學觀察 26
三、週邊單核細胞 (peripheral blood mononuclear cells, PBMC) 27
四、腸道形態學與細胞相變化 28
1. 腸道組織型態變化 28
2. 免疫組織化學染色 28
五、不同部位淋巴細胞族群的比例 28
六、B. fragilis對誘發不同部位淋巴細胞產生細胞素之影響 29
1. 經由LPS刺激 29
2. 經由ConA刺激 30
七、分析結直腸的細胞素與炎症基因表現量 33
第六章、討論 34
一、B. fragilis及接菌後之菌量變化 34
二、口服定殖B. fragilis菌對老鼠腸道形態學及相關淋巴組織的影響 34
三、流式細胞儀分析不同部位淋巴細胞族群的比例 36
四、B. fragilis對誘發不同部位淋巴細胞產生細胞素之影響 37
五、結直腸的細胞素與炎症基因表現量 38
第七章、結論 40
參考文獻 41

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