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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 陳明汝(Ming-Ju Chen) | |
dc.contributor.author | Wei-Sheng Hong | en |
dc.contributor.author | 洪偉盛 | zh_TW |
dc.date.accessioned | 2021-05-20T21:43:28Z | - |
dc.date.available | 2018-08-10 | |
dc.date.available | 2021-05-20T21:43:28Z | - |
dc.date.copyright | 2010-08-18 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-10 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10609 | - |
dc.description.abstract | 本研究由預備實驗結果得知,分離自蒙古組克弗爾上層液可刺激小鼠巨噬細胞RAW264.7細胞株產生TNF-α,其為前發炎反應相關之細胞激素,因此第二章試驗擬針對克弗爾免疫調節能力與克弗爾粒中單一乳酸菌菌株之關係進行研究,首先以小鼠巨噬細胞株RAW264.7 與小鼠腹水細胞為模式,觀察克弗爾上層液與單一菌株對細胞活化之情形,尋找刺激細胞激素分泌的最佳條件;同時研究引發免疫調節之上游機制,此部分試驗針對Toll-like receptor (TLR) 進行分析。試驗結果顯示,牛乳經克弗爾粒發酵24與48小時之克弗爾上層液可顯著提昇RAW264.7細胞株分泌前發炎反應細胞激素 (tumor necrosis factor (TNF)-α, interleukin (IL)-6以及IL-1β) 以及T helper (Th) 1細胞激素 (IL-12);可能的機能性成分為熱穩定性佳 (80℃加熱30分鐘仍具有刺激細胞激素之能力) 之物質。進一步比較四株自克弗爾粒分離的菌株,其免疫調節能力又以Lactobacillus kefiranofaciens M1及Lb. kefiri M2刺激之細胞激素分泌效果最為顯著,與牛乳克弗爾上層液的分泌量無顯著差異。免疫調節路徑方面,以TLR-2抗體針對RAW264.7細胞株進行試驗,顯示克弗爾上層液、單一菌株與其發酵上層液所刺激之細胞激素分泌量均顯著下降;進一步以TLR-2基因剔除鼠之腹水細胞進行實驗,結果顯示Lb. kefiranofaciens M1對於刺激其腹水細胞之IL-6分泌顯著下降,證實其免疫調節路徑系經由TLR-2。
第三章抗過敏之動物實驗。試驗中以克弗爾分離乳酸菌Lb. kefiranofaciens M1及Lb. kefiri M2進行試驗,首先利用小鼠脾臟及腹水細胞共同培養,刺激其分泌Th1與前發炎反應細胞激素,探討熱失活菌株 (85 ℃加熱30分鐘) 之免疫調節能力與活菌株與是否產生差異。進一步餵食Lb. kefiranofaciens M1於ovalbumin (OVA) 致敏小鼠,測定其Th1/Th2細胞激素、調節型T細胞之數量及血清中OVA-specific IgE之濃度。菌株樣品是以灌食方式直接與腸道接觸,因此試驗亦抽取腸道Peyer’s patch之RNA以進行小鼠全基因微陣列晶片分析,以瞭解菌株對於過敏小鼠其腸道黏膜系統所引起之整體基因變化。試驗證實熱失活之Lb. kefiranofaciens M1仍具有刺激脾臟與腹水細胞分泌細胞激素之能力,因此選用熱失活菌株進行動物試驗,結果顯示餵食菌株之OVA過敏小鼠,顯著提升脾臟細胞中Th1細胞激素 (IL-12),降低Th2細胞激素(IL-5)之分泌;同時提升脾臟中調節型T細胞之數量,而令其血中之OVA-specific IgE顯著降低。而RNA基因微陣列晶片分析之結果顯示,OVA過敏小鼠餵食菌株後,其Peyer’s patch之補體的基因表現俱顯著下降,同時顯著提升Ifnr、Cd2、Cd3、Cd28、Stat4 與Ccr7基因之表現。 經第三章動物試驗,已經證實克弗爾粒中之熱失活乳酸菌Lb. kefiranofaciens M1具有刺激分泌前發炎反應相關細胞激素之能力、提升Th1反應,改善OVA過敏小鼠之Th2傾向之因子。因此第四章試驗以第一型過敏反應之氣喘實驗動物模式進行,評估自克弗爾粒中分離之Lb. kefiranofaciens M1能否改善呼吸道之臨床症狀。實驗動物先以OVA進行腹腔注射外,再令其吸入OVA粉塵以產生氣喘,餵食不同濃度以及不同時間長度之樣品後,觀察以乙酰甲膽堿(methacholine) 引起之呼吸道阻力 (enhanced pause, Penh) 是否降低;測定脾臟與支氣管沖洗液中之Th2 (IL-4,IL-5與IL-13) 與Th17 (IL-17與IL-17F) 細胞激素之分泌量及血清中OVA-specific IgE之含量;並以組織切片觀察肺臟中免疫細胞浸潤與黏液分泌情況。結果顯示,以熱失活之Lb. kefiranofaciens M1餵食OVA氣喘小鼠,其餵食菌數達108 CFU以上,且以試驗全程每日皆餵食之方式,小鼠脾臟與支氣管沖洗液中之前發炎反應、Th2及Th17細胞激素顯著下降,並能夠顯著降低由乙酰甲膽堿所誘發的Penh 值;切片結果顯示肺臟切片中免疫細胞之浸潤以及黏液分泌顯著減少,血清中之OVA-specific IgE亦顯著下降。 | zh_TW |
dc.description.abstract | Kefir has long being considered good for health. Its health benefits include immunoregulatory effects. However, there is a lack of knowledge concerning the immunoregulatory effects induced by kefir lactic acid bacteria (LAB). In addition, the mechanisms responsible for these effects have not been fully determined. Thus, the objective of this study was to investigate the immunomodulating reaction and mechanisms of LAB isolated from kefir grains. Firstly, we investigated the in vitro immunomodulating capacity and mechanisms of kefir supernatants and kefir LAB by cytokine profiles through a toll-like receptor (TLR) pathway. Results demonstrated that kefir supernatants, obtained from kefir fermented more than 24 h, induced the production of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β) in RAW264.7 cells. Among four LAB isolated from kefir grains and their supernatants, Lactobacillus kefiranofaciens M1 and its supernatant had strong potential to induce in vitro production of TNF-α, IL-1β, IL-6 and T helper (Th) 1 (IL-12) cytokine in RAW264.7 cells and murine peritoneal macrophages. Moreover, blocking TLR-2 using anti-TLR-2 mAb and TLR-2-/- mice showed a significant inhibition of cytokine production. These findings indicated that kefir influenced the secretion cytokines through TLR-2.
According to the above results, the LAB may be beneficial for the promotion of Th1/Th2 balances and alleviating of type 1 hypersensitive response. In this context, we examined the anti-allergic effects of Lb. kefiranofaciens M1 and Lb. kefiri M2 isolated from kefir grains by modulation of Th1/Th2 balances and inhibition of immunoglobulin (Ig) E production in ovalbumin (OVA)-sensitized Th2-polarized mice. This study demonstrated that oral feeding of heat inactivated Lb. kefiranofaciens M1 from kefir grains effectively inhibited Ig E production in response to OVA in vivo. The pattern of cytokine production by splenocyte cells revealed that the levels of cytokines produced by Th 1 cells increased, and those of cytokines produced by Th2 cells decreased in the heat inactivated M1 feeding group. These findings indicated that Lb. kefiranofaciens M1 in the kefir played an important role in anti-allergic activities. By additional analysis using flow cytometry and microarray, the mechanism of suppression of IgE production by oral feeding of the heat inactivated M1 probably occurs because of up-regulation of the expression of Cd2, Stat4, and Ifnr leading to skewing the Th1/Th2 balance toward Th1 dominance, elevation of the CD4+CD25+ regulatory T (Treg) cells percentage and reduction of activated CD19+ B cells. Down-regulation of complement system and components was also involved in suppression of IgE production. Further studies of different type 1 hypersensitivity diseases were necessary to address the anti-allergic effects of lactobacilli isolated from kefir grains. Allergic asthma, a type 1 hypersensitivity, is characterized by allergen-induced chronic inflammation of the lungs and airway hyperresponsiveness (AHR), associated with the enhancement of allergen-induced eosinophilia, goblet cell hyperplasia, allergen-specific IgE levels and Th2 dominant cytokines. In the third part of this study, we assessed the anti-asthmatic effects of Lb. kefiranofaciens M1 and its fermented milk in different feeding procedures and dosages. The cellular mechanisms of Lb. kefiranofaciens M1 in anti-allegic asthmatic effects were also evaluated. The mice oral administrated M1 sample strongly inhibited the production of Th2 (IL-4, IL-5 and IL-13), proinflammatory (IL-6, IL-1β, TNF-α and CCL20) and Th17 cytokines in splenocytes and bronchoalveolar fluid (BAL) in the OVA-allergic asthma mice in a timing of the intervention and a dose dependent manner. The increase in regulatory T cell population for oral administrated M1 in splenocytes in the allergic asthma mice was also observed. Additionally, all features of the asthmatic phenotype, including specific IgE production, airway inflammation, and development of airway hyperresponsiveness (AHR), were depressed in a dose dependent manner. Taken together, these findings indicate a possibility that the intake of kefir lactobacilli, Lb. kefiranofaciens M1, may be effective in alleviating asthmatic symptoms. Both the exposure time and the dosage are two important factors affecting the anti-allergic asthmatic effectiveness of Lb. kefiranofaciens M1. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T21:43:28Z (GMT). No. of bitstreams: 1 ntu-99-D94626003-1.pdf: 6128653 bytes, checksum: 8546770a4238dc568b242a8bc32ab7a6 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要 i
英文摘要 iii 研究背景 1 第一章:文獻整理 2 一、免疫調節之介紹 2 (一) 細胞激素(cytokine) 2 1. T輔助細胞第一型與第二型 2 2. T輔助細胞第17型 3 3. 調節型T細胞 (Regulatory T cell, Treg cell) 4 (二) 第一型過敏反應 (Type I hypersensitivity reaction) 5 (三) 氣喘 (Asthma) 6 (四) 補體 (complement) 7 二、乳酸菌與免疫調節 14 (一) 周邊血液單核球細胞 (peripheral blood mononuclear cells, MNCs) 14 1. T細胞 (T lymphocytes) 14 2. 調節型T細胞 (Regulatory T cell, Treg cell) 15 3. 自然殺手細胞 (natural killer cells, NK cells) 16 4. 周邊血液單核球細胞 (peripheral blood mononuclear cells, MNCs) 16 (二) 抗原呈現細胞 (Antigen-presenting cells, APC)` 17 (三) 次級淋巴組織 18 1. 脾臟 18 2. 腸道淋巴組織 19 (四) 乳酸菌與第一型過敏反應 20 (五) 乳酸菌與氣喘 20 (六) 乳酸菌之免疫調節物質 21 三、克弗爾發酵乳與免疫調節 25 (一) 餵食實驗動物以探討克弗爾之功效 25 (二) 藉由致病動物探討克弗爾改善免疫疾病之功效 25 四、本研究室在克弗爾發酵乳之研究成果 27 (一) 新竹克弗爾發酵乳機能性研究 27 (二) 克弗爾粒中之菌株分離鑑別 28 預備實驗 29 實驗動機及重要性 31 總試驗流程 32 第二章:探討克弗爾上層液與自克弗爾粒分離之乳酸菌經由Toll-like receptor 刺激巨噬細胞分泌細胞激素之功效 34 一、摘要 34 二、材料與方法 35 (一) 試驗流程 35 (二) 克弗爾樣品之製備 35 (三) 乳酸菌之菌種來源、培養與製備 36 (四) 克弗爾與RAW264.7細胞株共培養 36 (五) 克弗爾與小鼠巨噬細胞共培養 37 (六) 細胞激素測定 37 (七) 乳酸菌中免疫物質特性分析 37 (八) Blocking試驗以確認克弗爾活化之路徑 38 (九) 統計分析 38 三、結果 39 (一) 克弗爾上層液對於RAW264.7細胞株分泌細胞激素之影響 39 (二) 克弗爾中乳酸菌與其發酵乳上層液對於RAW264.7細胞株分泌細胞 激素之影響 39 (三) 探討克弗爾中具有機能性成分 45 (四) 探討免疫活化之受器 45 四、討論 51 第三章:探討自克弗爾粒分離之乳酸桿菌之抗過敏功效 55 一、摘要 55 二、材料與方法 57 (一) 試驗流程 57 (二) 乳酸菌之菌種來源、培養與製備 57 (三) 體外細胞激素試驗小鼠巨噬細胞與脾臟細胞 57 (四) 致敏小鼠動物實驗 59 (五) 細胞激素測定 60 (六) Microarray 60 (七) 統計分析 61 三、結果 62 (一) 自克弗爾分離之乳酸桿菌對於巨噬細胞與脾臟細胞細胞激素分泌之 影響 62 (二) 餵食熱失活Lb. kefiranofaciens M1對於IgE與OVA-specific IgE分泌 之影響 62 (三) 餵食熱失活Lb. kefiranofaciens M1對於致敏小鼠脾臟細胞激素之 影響 67 (四) 餵食熱失活Lb. kefiranofaciens M1對於致敏小鼠脾臟中CD4+CD25+ 調節型T細胞與CD19+ B細胞之影響 67 (五) 餵食熱失活Lb. kefiranofaciens M1 對於調控致敏小鼠Peyer’s patch 基因表現之影響 67 四、討論 72 第四章:探討自克弗爾粒分離之熱失活Lb. kefiranofaciens M1與其發酵乳 改善過敏性氣喘反應之功效 75 一、摘要 75 二、材料與方法 76 (一) 試驗流程 76 (二) 乳酸菌之製備與處理 76 (三) 氣喘小鼠之致敏與抗原吸入 76 (四) 乳酸菌餵食之不同處理組 77 (五) 呼吸道阻力測試 77 (六) 支氣管沖洗液之取得與肺臟切片 78 (七) 脾臟細胞之分離與CD4+CD25+調節型T細胞之分析 78 (八) 血清 79 (九) 細胞激素測定 79 (十) 統計分析 79 三、結果 80 (一) 熱失活Lb. kefiranofaciens M1以不同餵食處理對於氣喘小鼠之影響 80 (二) 不同劑量之Lb. kefiranofaciens M1對於氣喘小鼠脾臟與之支氣管沖 洗液之細胞激素分泌之影響 80 (三) 不同劑量之Lb. kefiranofaciens M1對於氣喘小鼠血清中OVA-specific IgE之影響 81 (四) 不同劑量之Lb. kefiranofaciens M1對於氣喘小鼠肺臟切片之影響 81 (五) Lb. kefiranofaciens M1對於氣喘小鼠肺功能之影響 81 四、討論 91 第五章:結論 95 參考文獻 97 附錄 117 | |
dc.language.iso | zh-TW | |
dc.title | 自克弗爾分離乳酸菌於第一型過敏反應之研究 | zh_TW |
dc.title | Investigation of Lactic Acid Bacteria Isolated from Kefir Grains on Type I Hypersensitivity | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 林慶文,王政騰,黃英豪,蔡英傑,廖啟成,潘子明 | |
dc.subject.keyword | 克弗爾,第一型過敏反應,氣喘,細胞激素,乳酸菌, | zh_TW |
dc.subject.keyword | asthma,cytokine,lactic acid bacteria,kefir,type 1 hypersensitivity, | en |
dc.relation.page | 116 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2010-08-10 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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