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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林璧鳳(Bi-Fong Lin) | |
| dc.contributor.author | Yi-Ting Tsai | en |
| dc.contributor.author | 蔡依廷 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:24:10Z | - |
| dc.date.available | 2014-08-20 | |
| dc.date.copyright | 2009-08-20 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-17 | |
| dc.identifier.citation | 謝貴雄 (1995) 小兒氣喘之預防和處理。中華民國兒科醫學會雜誌，36 : S9-20
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43601 | - |
| dc.description.abstract | 口服耐受性是以口服抗原的方式，使個體再度接觸到抗原時傾向產生免疫耐受性，且多數口服蛋白對個體不產生毒性，所誘發的免疫調節具有抗原專一性，具應用到過敏性疾病治療的潛力。本實驗室之前研究發現，給予塵蟎蛋白 (Dp 2)致敏小鼠管餵高劑量 Dp 2 一週，可有效誘發口服耐受性。此外，研究指出，維生素會影響免疫反應，故本實驗欲探討葉酸對口服塵蟎蛋白的氣喘小鼠口服耐受性的影響。
本研究第一階段給予致敏小鼠口服低、高劑量 (100 μg、500 μg) 的 Dp 2 蛋白8 天，再補充 10 倍葉酸 2 至 8 週，探討葉酸對誘發氣喘小鼠口服耐受性所需口服抗原劑量的影響。結果顯示，2 週短期補充葉酸顯著增加小鼠脾臟抗原特異性 IFN-γ 分泌，和增加 MLN IL-2 分泌的趨勢，而顯著抑制 IL-4 的分泌量。8 週的葉酸補充，並追加 3 次致敏後犧牲小鼠，發現葉酸顯著提昇小鼠肺氣管沖洗液 (BALF) 中 IL-2 的含量，且有增加 Peyer’s patch 中 CD4+CD25+Foxp3＋ 調節性 T 細胞比例的趨勢，顯示補充 10 倍葉酸有利於口服耐受性的誘發，調控 Dp 2 致敏的氣喘小鼠體內免疫反應更為傾向 Th1 反應。高劑量 Dp 2 雖然增加小鼠血清 IgE 濃度，但顯著降低 BALF 中嗜酸性白血球比例與脾臟 Th2 細胞激素的分泌，因此口服耐受性的誘發仍以 500 μg Dp 2 較可行。 第二階段研究則進一步探討補充葉酸的含量對口服耐受性的影響。給予致敏小鼠口服 500 μg Dp 2 蛋白 8 天，並同時給予含有 0、1、3、5 及 10 倍葉酸的飼料 8 週，同時追加 3 次致敏後犧牲小鼠，分析各項免疫指標。結果顯示，補充 5 倍葉酸顯著降低小鼠脾臟 Th1 細胞激素分泌與特異性增生能力，並降低 MLN 特異性 IL-5 的分泌；補充 10 倍葉酸顯著抑制脾臟細胞 Th1 細胞激素、IL-10、TGF-β 的分泌，也顯著增加脾臟 CD4+CD25+Foxp3+ 調節性 T 細胞比例；缺乏葉酸顯著增加 MLN 抗原特異性 IL-2 分泌，但顯著降低脾臟與 MLN 中 CD4+CD25+Foxp3+、CD4+Foxp3+ 調節性 T 細胞比例與表現強度。 由本實驗結果得知，Dp 2 致敏小鼠氣喘模式中，口服 Dp 2同時補充 10 倍葉酸，有助於誘發口服耐受性。短期的葉酸補充，透過調控腸道免疫組織影響個體免疫反應傾向 Th1；長期葉酸補充，顯著增加脾臟 CD4+CD25+Foxp3+ 調節性 T 細胞比例。葉酸的缺乏則降低 Foxp3 表現強度，不利口服耐受性的維持。 | zh_TW |
| dc.description.abstract | Oral tolerance is a state of immunological unresponsiveness to antigen induced by feeding. Most oral antigens are non-toxic proteins, and their ability of inducing antigen-specific immune regulation has made oral tolerance a highly potential treatment to allergy susceptible individuals. We had successfully induced oral tolerance in dust mite protein (Dp 2)-sensitized BALB/c mice with high dose (500 μg) Dp 2 oral administration for 1 week. In this study, we showed the effect of water-soluble vitamin folate on asthma mice oral administered with Dp 2.
To investigate if folate affects the antigen dose to induce oral tolerance, we fed the Dp2-sensitized mice with a diet containing 10 times the folate requirement, and gave the mice high or low dose (500 μg and 100 μg) Dp 2 for 8 days. After 2 weeks of short-term folate supplement, there was a trend toward an increase of mesenteric lymph node (MLN) IL-2 secretion. Also, the short-term folate supplement significantly increased splenocyte-specific IFN-γ secretion, while decreased the MLN IL-4 secretion. Subsequently, after 3 intra-peritoneal boosts, 8 weeks of long-term folate supplement significantly increased bronchoalveolar lavage fluid (BALF) IL-2 and slightly increased CD4+CD25+Foxp3＋ regulatory T cell (Treg) percentage in Peyer’s patch. These results indicate that folate plays a role in shifting immune response toward Th1 in asthma mice. Though high dose Dp 2 increased serum IgE level, it significantly reduced eosinophil percentage in BALF and spleen Th2 cytokine secretion. Therefore, oral tolerance induction remained more effective when using high dose of Dp 2. In the following experiment, we demonstrated the effect of folate contents on tolerized mice. Dp 2-sensitized mice were given high dose Dp 2 for 8 days and diets containing 0, 1, 3, 5, 10 times folate for 8 weeks with 3 intra-peritoneal boosts. 5x folate supplement reduced splenocyte Th1 cytokine secretions as well as antigen-specific proliferation and MLN IL-5 secretion. 10x supplement significantly decreased splenocyte Th1 cytokines, IL-10, and TGF-β while increased CD4+CD25+Foxp3+ Treg percentage in spleen. Folate-deficient diet reduced CD4+CD25+Foxp3+ and CD4+Foxp3+ Treg percentage and Foxp3 expression intensity in both spleen and MLN, but increased antigen-specific IL-2 secretion in MLN. Our data show that 10x folate supplement enhances oral tolerance induction by high dose Dp 2 in Dp 2-sensitized mice. Short-term folate supplement tends to shift immune reaction toward Th1 through mucosa while long-term folate supplement increases spleen CD4+CD25+Foxp3+ Treg percentage. Folate deficiency decreses Foxp3 intensity and thus impairs oral tolerance maintenance. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:24:10Z (GMT). No. of bitstreams: 1 ntu-98-R96b47307-1.pdf: 10497306 bytes, checksum: 3b5031da854b24038e0c3b2139a7f9f9 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 中文摘要………………………………………………………………………………....I
英文摘要……………………………………………………………………………….III 目錄……………………………………………………………………………………..V 表目錄…………………………………………………………………………………VII 圖目錄…………………………………………………………………………………..X 第一章 序論…………………………………………………………………………….1 第一節 文獻回顧 1 一、過敏性氣喘 1 (一) 簡介與定義 1 (二) 過敏性氣喘的致病機轉 2 (三) 過敏性氣喘的預防與治療方法 3 二、塵蟎過敏原 Dp 2 6 (一) 塵蟎簡介 6 (二) Dp 2 6 三、口服耐受性 7 (一) 簡介與定義 7 (二) 誘發口服耐受性的可能機制 8 (三) 營養素對耐受性的影響 10 (四) 口服耐受性在治療疾病上的應用 11 四、葉酸 13 (一) 葉酸簡介 13 (二) 葉酸與免疫的關係 14 第二節 研究動機 16 第二章 實驗材料與分析方法…………………………………………………………17 第一節 Dp 2 蛋白之生產與純化 17 一、來源 17 二、菌種解凍活化、菌種準備 17 三、蛋白質大量生產 17 四、蛋白質回收與純化 19 五、蛋白質分析 19 第二節 動物實驗設計 23 一、動物飼養流程與實驗分組 23 (一) 飲食高量葉酸對口服不同 Dp2 劑量致敏 BALB/c 小鼠誘發口服耐受性的影響 (實驗一) 23 (二) 飲食葉酸對 Dp2 致敏後誘發口服耐受性 BALB/c 小鼠的影響 (實驗二) 26 二、致敏方式 28 三、氣管內刺激 (intra-tracheal challenge, i.t.) 28 四、口服耐受性的建立 — 餵食 Dp 2 蛋白 29 第三節 動物犧牲及實驗材料收集與分析 29 一、血清樣品的收集 29 二、呼吸道過度反應 (airway hyperresponsiveness, AHR) 測定 30 三、動物犧牲 30 四、肺沖洗液及肺部細胞的取得 31 五、脾臟細胞的取得與培養 31 六、脾臟細胞增生測定 32 七、腸繫膜淋巴結細胞取得與培養 33 八、皮耶氏體細胞的取得與培養 34 九、脾臟、腸繫膜淋巴結、皮耶氏體細胞表面標記與 Foxp3 轉錄因子分析 34 十、血清 Dp2 特異性抗體含量分析 36 十一、血清非特異性抗體含量分析 37 十二、細胞激素分泌量測定 38 第四節 統計分析方法 40 第三章 結果……………………………………………………………………………41 第一節 飲食葉酸對口服不同 Dp 劑量的致敏小鼠口服耐受性誘發的影響 41 一、短期補充葉酸 41 (一) 生長情形與攝食狀況 41 (二) 器官重量、相對器官重量與血清葉酸濃度 41 (三) 血清中 Dp 2 特異性抗體與總抗體含量 41 (四) 脾臟細胞細胞激素分泌量 42 (五)腸繫膜淋巴結細胞細胞激素分泌量 42 (六) 腸繫膜淋巴結細胞及皮耶氏體細胞 Foxp3 轉錄因子的表現 43 二、長期補充葉酸 57 (一) 生長情形與攝食狀況 57 (二) 器官重量與、相對器官重量與血清葉酸濃度 57 (三) 肺氣管沖洗液 (BALF) 中細胞激素含量 57 (四) 肺氣管沖洗液中各種細胞數目 57 (五) 血清中總抗體及 Dp 2 特異性抗體含量 58 (六) 脾臟細胞、腸繫膜淋巴結細胞細胞激素分泌量 58 (七) 腸繫膜淋巴結細胞及皮耶氏體細胞 Foxp3 轉錄因子的表現 58 (八) 脾臟細胞增生能力 59 第二節 飲食葉酸含量對 Dp 2 致敏 BALB/c 小鼠誘發耐受性的影響 76 一、生長情形與攝食狀況 76 二、器官重量與相對器官重量 76 三、血清葉酸濃度 77 四、呼吸道過度反應 (AHR) 77 五、肺氣管沖洗液 (BALF) 中細胞激素含量 77 六、肺氣管沖洗液中各種細胞數目 77 七、血清中 Dp 2 特異性抗體與總抗體含量 78 八、脾臟細胞細胞激素分泌量 78 九、腸繫膜淋巴結細胞細胞激素分泌 79 十、 脾臟細胞、腸繫膜淋巴結細胞及皮耶氏體細胞 Foxp3 轉錄因子的表現 80 十一、脾臟細胞增生能力 80 第四章 討論與總結…………………………………………………………………..101 第一節 討論 101 一、葉酸對小鼠生長的影響 101 二、葉酸對肺部免疫指標的影響 101 三、葉酸對血清抗體的影響 102 四、葉酸對細胞激素分泌的影響 103 五、葉酸對 Foxp3 轉錄因子的影響 104 六、葉酸對脾臟細胞增生的影響 105 第二節 綜合討論 106 一、口服耐受性的誘發與全身性的免疫反應 106 二、葉酸營養狀況對本實驗的影響 107 三、短期與長期補充葉酸的影響比較 108 四、Treg 在的重要性 110 五、蛋白質純化程度對實驗的影響 111 六、小鼠死亡因素的探討及本實驗需改善之處 112 第三節 總結 112 參考文獻.……………………………………………………………...……………...116 表目錄 表 1.1 各種調節性 T 細胞 9 表 2.1 實驗一使用之飼料組成 25 表 2.2 實驗一各組小鼠餵食飼料、致敏、刺激及管餵成分表 25 表 2.3 實驗二使用之飼料組成 27 表 2.4 實驗二各組小鼠餵食飼料、致敏、刺激及管餵成分表 27 表 3.1 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠生長與攝食的影響 44 表 3.2 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠器官重量與相對器官重的影響 45 表 3.3 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠血清 Dp2 特異性抗體含量的影響 47 表 3.4 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠血清總抗體含量的影響 48 表 3.5 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠脾臟細胞 Th1 細胞激素分泌量的影響 49 表 3.6 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠脾臟細胞 Th2 細胞激素分泌量的影響 50 表 3.7 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠脾臟細胞 IL-10、TGF-β 細胞激素分泌量的影響 51 表 3.8 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠腸繫膜淋巴結細胞 Th1 細胞激素分泌量的影響 52 表 3.9 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠腸繫膜淋巴結細胞 Th2 細胞激素分泌量的影響 53 表 3.10 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠腸繫膜淋巴結細胞 IL-10 、TGF-β 細胞激素分泌量的影響 54 表 3.11 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠生長情形與攝食的影響 60 表 3.12 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠器官重量與相對器官重的影響 61 表 3.13 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠 BALF 中細胞激素與趨化激素的影響 63 表 3.14 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠血清 Dp2 特異性抗體含量的影響 65 表 3.15 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠血清總抗體含量的影響 66 表 3.16 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠脾臟細胞 Th1 細胞激素分泌量的影響 67 表 3.17 長期攝食高量 葉酸對口服不同 Dp 2 劑量致敏小鼠脾臟細胞 Th2 細胞激素分泌量的影響 68 表 3.18 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠脾臟細胞 IL-10、TGF-β 細胞激素分泌量的影響 69 表 3.19 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠腸繫膜淋巴結細胞 Th1 細胞激素分泌量的影響 70 表 3.20 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠腸繫膜淋巴結細胞 Th2 細胞激素分泌量的影響 71 表 3.21 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠腸繫膜淋巴結細胞 IL-10、TGF-β 細胞激素分泌量的影響 72 表 3.22 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠攝食的影響 82 表 3.23 飲食葉酸對 Dp 2 致敏後誘發口服耐受性之 BALB/c 小鼠器官重量與相對器官重量的影響 83 表 3.24 飲食葉酸對 Dp 2 致敏後誘發口服耐受性之 BALB/c 小鼠 BALF 中細胞激素與趨化激素含量的影響 86 表 3.25 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠脾臟細胞 Th1 細胞激素分泌量的影響 90 表 3.26 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠脾臟細胞 Th2 細胞激素分泌量的影響 91 表 3.27 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠脾臟細胞 IL-10、TGF-β 細胞激素分泌量的影響 92 表 3.28 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠腸繫膜淋巴結細胞 Th1 細胞激素分泌量的影響 93 表 3.29 飲食葉酸對 Dp 2 致敏後誘口服發耐受性 BALB/c 小鼠腸繫膜淋巴結細胞 Th2 細胞激素分泌量的影響 94 表 3.30 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠腸繫膜淋巴結細胞 IL-10、TGF-β 細胞激素分泌量的影響 95 表 3.31 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠腸繫膜淋巴結細胞激素 Th2/Th1 ratio 的影響 96 表 3.32 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠脾臟細胞增生能力的影響 100 表 4.1 補充葉酸對本實驗小鼠各項免疫指標的影響 114 圖目錄 圖 1.1 (A) 葉酸的結構 (B) 葉酸的其他形式 15 圖 1.2 葉酸涉及的單碳代謝 16 圖 3.1 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠血清葉酸含量的影響 46 圖 3.2 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠腸繫膜淋巴結細胞 Foxp3 轉錄因子表現的影響 55 圖 3.3 短期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠皮耶氏體細胞 Foxp3 轉錄因子表現的影響 56 圖 3.4 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠血清葉酸含量的影響 62 圖 3.5 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠肺沖洗液細胞種類的影響 64 圖 3.6 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠腸繫膜淋巴結細胞 Foxp3 轉錄因子表現的影響 73 圖 3.7 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠皮耶氏體細胞 Foxp3 轉錄因子表現的影響 74 圖 3.8 長期攝食高量葉酸對口服不同 Dp 2 劑量致敏小鼠脾臟細胞增生能力的影響 75 圖3.9 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠生長的影響 81 圖 3.10 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠血清葉酸濃度的影響 84 圖 3.11 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠呼吸道過度反應的影響 85 圖 3.12 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠肺沖洗液細胞種類的影響 87 圖 3.13 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠血清 Dp 2 蛋白特異性抗體含量的影響 88 圖 3.14 飲食葉酸對 Dp 2 致敏後誘發口服耐受性之 BALB/c 小鼠血清總抗體含量的影響 89 圖 3.15 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠脾臟細胞 Foxp3 轉錄因子表現的影響 97 圖 3.16 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠腸繫膜淋巴結細胞 Foxp3 轉錄因子表現的影響 98 圖 3.17 飲食葉酸對 Dp 2 致敏後誘發口服耐受性 BALB/c 小鼠皮耶氏體細胞 Foxp3 轉錄因子表現的影響 99 圖 4.1 本實驗純化之蛋白質的 Western blot 結果 115 | |
| dc.language.iso | zh-TW | |
| dc.title | 葉酸對塵螨蛋白致敏 BALB/c 小鼠口服耐受性的影響 | zh_TW |
| dc.title | Effect of dietary folate supplement on oral tolerance
development in Dp2-sensitized BALB/c mice | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 江伯倫,李昆達,吳文勉,許瑞芬 | |
| dc.subject.keyword | 口服耐受性,葉酸,塵螨,蛋白,MLN,Foxp3+調節性T細胞, | zh_TW |
| dc.subject.keyword | oral tolerance,folate,Dp 2,MLN,Peyer’s patch,Foxp3+ Treg, | en |
| dc.relation.page | 126 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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