江伯倫臺灣大學：口腔生物科學研究所游詩怡Yu, Shih-YiShih-YiYu2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51283「口服耐受性」(oral tolerance)是一種利用口服抗原以降低抗原特異性免疫反應的機制，此特殊現象期望可應用至臨床過敏疾病的治療，而過敏性氣喘即為一種過度免疫反應且具慢性呼吸道發炎病徵的普遍疾病。此研究中，我們利用OVA-TCR (OVA-T-cell receptor)轉殖基因鼠建立過敏性氣喘之動物模式，而 OVA-TCR能專一辨認卵蛋白(ovalbumin, OVA)上第323-339的胜肽片段，故能便利於免疫機轉的探討。首先，本實驗於小鼠中成功誘發了呼吸道過敏、大量OVA特異性IgE抗體生成、及嗜酸性白血球(eosinophil)不正常聚集等現象；致敏前，同以連續管餵小鼠OVA抗原(0.5mg/day) 5天或10天後，結果顯示皆能緩和呼吸道阻力、減低肺部發炎細胞浸潤現象、降低血清中OVA特異性IgE抗體的生成量、也減少肺部沖洗液及脾臟細胞內第二型輔助T細胞(type II helper T cell, Th2)細胞激素的分泌(IL-4、IL-5)。甚之，在口服OVA抗原的小鼠脾臟中，體外以該抗原刺激後加以培養，仍能抑制脾臟淋巴細胞的增生能力。綜合以上，口服低劑量抗原的小鼠有效抑制了局部和全身性Th2免疫反應，進而調節B細胞的同型轉換(class switch)使IgE抗體生成減少，並減緩過敏性呼吸道發炎之現象。 繼之，口服OVA抗原誘發免疫耐受的現象之後，欲進一步探討OVA-TCR轉殖基因小鼠腸道淋巴皮耶式體(Peyer’s Patch, PP)中基因的表現，以Affymetrix Mouse GeneChip取得初步基因資料。實驗中小鼠連續口服OVA抗原(200mg/day、20mg/day、0.5mg/day)達5天後，犧牲分別取得皮耶式體、腸繫膜淋巴組織(mesenteric lymph nodes, MLN)、及脾臟細胞分選出的CD4+CD25+ T細胞，再利用real-time PCR加以分析初步資料中各高度表現的基因。實驗結果顯示，基因foxp3、crem、satb等轉錄因子高度表現於有餵食OVA抗原小鼠的皮耶式體中；皮耶式體與脾臟細胞分選而得的CD4+CD25+ T細胞內，觀察到與抑制免疫細胞、調節使反應低落的基因rbm10、mterfd1等，以及其他免疫調節相關基因cd226、crip3亦於皮耶式體與腸繫膜淋巴組織中，經餵食OVA抗原後皆顯著提高了表現量；而實驗亦利用免疫細胞株作基因表現位置的確認。有趣的是，以上免疫耐受相關基因大多高度表現於皮耶式體的B細胞中；然而，於其他文獻中已知，未經活化的B細胞是一種效能不佳的抗原呈獻細胞(antigen-presenting cells, APC)，無法啟動T細胞活化的機能，更使T細胞在體外轉而發育為調控型T細胞，並具抑制免疫反應的功能。因此，本實驗推論高度表現於皮耶式體B細胞內與口服耐受機制相關的基因們，可能在抑制過度免疫反應上佔有一席調節的角色。Oral tolerance is defined as a state of immunological unresponsiveness to a specific antigen after exposure to that antigen via the enteric route. This characteristic phenomenon might be used as a potential treatment for allergic diseases such as asthma. Asthma is usually regarded as a hyper-responsive airway disease involving chronic inflammation. First, we established an animal model using OVA-T-cell receptor (OVA-TCR) transgenic mice with airway hyperresponsiveness, antigen-specific IgE production, and eosinophilia. The OVA-TCR can recognize the 323-339 peptide fragment of ovalbumin (OVA). In this study, oral tolerance was induced by tube-feeding transgenic mice with 0.5mg/day OVA protein daily, simultaneously sensitized by OVA protein with aluminum hydroxide. Oral-administered OVA protein (0.5mg/day for 5 or 10 consecutive days) resulted in significant lowered airway hypersensitivity as well as reduced inflammative cells infiltration, titer of serum OVA-specific IgE, IL-5 secretion in bronchoalveolar lavage fluid(BALF), and IL-4 secretion in splenocyte. Furthermore, the proliferation of OVA-specific splenocyte was inhibited by oral-administered OVA protein. Taken together, oral administration of low-dose OVA protein induced oral tolerance, inhibited both local and systemic type II helper T cell (Th2) response, and significantly relieved airway allergic hypersensitivity. The second goal of the present study was to evaluate the Peyer's Patch (PP) gene expression in OVA-TCR transgenic mice after oral administration of OVA protein to induce specific immune tolerance. PP gene expression patterns were examined with Affymetrix Mouse GeneChip and further confirmed by real-time PCR. We detected the gene expression level on PP tissues, mesenteric lymph nodes (MLN), and CD4+CD25+ T cells derived from spleen after oral feeding of OVA protein (200mg/day, 20mg/day, and 0.5mg/day for 5 consecutive days). The results showed that transcription factor-associated genes, including foxp3, crem, and satb1, were highly expressed by the PP of the OVA-fed mice, and the growth suppression- and anergy- associated genes rbm10 and mterfd1 were elevated both in PP and CD4+CD25+ T cells derived from spleen after oral feeding of OVA protein. In addition to these genes, cd226 and crip3 were also up-regulated on PP and MLN upon OVA protein administration. Interestingly, most of these genes were mainly expressed in the B cells of PP. Moreover, as shown in some studies, naïve B cells are ineffective antigen-presenting cells (APC) and considered unable to activate naïve T cells, and even induced regulatory T cells inhibiting priming of fresh T cells in a contact-dependent manner in vitro. These results suggested that genes mediating oral tolerance were highly expressed in B cells of PP and may have a special mechanism to regulate immune tolerance.口試委員會審定書 誌謝…………………………………………………………………………………... I 中文摘要……………………………………………………………………………III 英文摘要……………………………………………………………………………….V 目錄……………………………………………………………………………………VII 圖表目錄………………………………………………………………………………X 第壹章 文獻回顧……………………………………………………………………1 第一節 過敏性氣喘………………………………………………………….....1 1.1.1 簡介……………………………………………………………….....1 1.1.2 氣喘的病理現象……………………………………………………..2 1.1.3 氣喘疾病致病之機轉………………………………………………..3 1.1.4 目前治療氣喘之方式………………………………………………4 第二節 腸道黏膜性免疫系統………………………………………………….6 1.2.1 簡介…………………………………………………………………6 1.2.2 腸道免疫作用………………………………………………………...6 第三節 口服耐受性……………………………………………………………7 1.3.1 簡介…………………………………………………………………7 1.3.2 口服耐受性之可能機轉……………………………………………8 1.3.3 利用口服耐受性治療免疫疾病之應用……………………………9 第貳章 研究目的……………………………………………………………………11 第參章 實驗材料與方法…………………………………………………………….12 第一節 引發口服耐受機制對於過敏性氣喘小鼠之免疫影響………………12 3.1.1 實驗試劑與配製……………………………………………………12 3.1.2 過敏性氣喘模式動物之建立……………………………………….14 3.1.3 口服耐受機制之建立……………………………………………….15 3.1.4 呼吸道過敏反應之測定…………………………………………….16 3.1.5 實驗動物犧牲……………………………………………………….16 3.1.6 各項免疫功能之測定……………………………………………….19 3.1.7 統計方法……………………………………………………………23 第二節 黏膜淋巴組織中口服耐受機制相關基因之偵測……………………24 3.2.1 實驗試劑與配製……………………………………………………24 3.2.2 口服耐受機制之建立………………………………………………24 3.2.3 實驗動物犧牲………………………………………………………25 3.2.4 口服耐受機制相關基因之偵測……………………………………30 第三節 口服耐受機制與相關免疫細胞的基因表現………………………….33 3.3.1 各項細胞株之培養與活化………………………………………….33 3.3.2 利用流式細胞計數儀分析特定細胞族…………………………….34 3.3.3 口服耐受機制相關基因之測定……………………………………34 第肆章 實驗結果……………………………………………………………………36 第一節 引發口服耐受機制對於過敏性氣喘小鼠之免疫影響……………….36 4.1.1 呼吸道過敏反應……………………………………………………36 4.1.2 血清中OVA特異性IgE抗體……………………………………..37 4.1.3 肺部沖洗液細胞族群組成、及細胞激素分泌量…………………37 4.1.4 巴細胞增生能力...............................................................................38 4.1.5 腸道黏膜淋巴組織：皮耶式體、腸繫膜淋巴組織初代培養細胞激素之分泌量…………………………………………………………38 4.1.6 脾臟細胞初代培養細胞激素分泌量………………………………39 4.1.7 皮耶式體淋巴組織中調控型細胞激素之分泌探討………………39 4.1.8 腸繫膜淋巴組織中調控型細胞激素之分泌探討…………………40 4.1.9 脾臟細胞中調控型細胞激素之分泌探討…………………………41 第二節 黏膜淋巴組織中口服耐受機制相關基因之偵測…………………….42 4.2.1 皮耶式體淋巴組織中口服耐受機制相關基因的表現…………….42 4.2.2 CD4+CD25+ T細胞中口服耐受機制相關基因的表現……………43 4.2.3 於皮耶式體、腸繫膜淋巴組織各類免疫細胞中口服耐受機制相關基因的表現………………………………………………………….44 第三節 口服耐受機制與相關免疫細胞的基因表現………………………46 4.3.1 活化各免疫細胞後細胞激素與特定細胞表面分子的表現量……46 4.3.2 口服耐受機制與相關活化免疫細胞的基因表現…………………47 第伍章 討論與展望………………………………………………………………….48 第一節 引發口服耐受機制對於過敏性氣喘小鼠之免疫影響………………48 5.1.1 過敏性氣喘模式動物之建立………………………………………48 5.1.2 口服耐受機制之建立………………………………………………49 5.1.3 口服低劑量抗原對於過敏性氣喘小鼠肺部細胞激素分泌之影響.50 5.1.4 口服低劑量抗原對於過敏性氣喘小鼠腸道局部免疫反應之影響.51 5.1.5 短期口服抗原對於過敏性氣喘小鼠全身性免疫反應之影響……52 第二節 黏膜淋巴組織、及相關免疫細胞中口服耐受機制相關基因之合併討 論……………………………………………………………………..54 5.2.1 口服耐受機制相關基因於黏膜淋巴組織內表現與機制之探討…54 圖表…………………………………………………………………………………..58 參考文獻…………………………………………………………………….……95 附錄……………………………………………………………………………….1041099620 bytesapplication/pdfen-US口服耐受腸道黏膜免疫皮耶式體腸繫膜淋巴調控型T細胞oral tolerancemucosal immune systemmesenteric lymph nodesregulatory T cellotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51283/1/ntu-96-R94450006-1.pdf