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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 潘子明 | |
dc.contributor.author | Yueh-Ting Tsai | en |
dc.contributor.author | 蔡岳廷 | zh_TW |
dc.date.accessioned | 2021-06-15T06:47:25Z | - |
dc.date.available | 2014-07-07 | |
dc.date.copyright | 2011-07-07 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-06-04 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48150 | - |
dc.description.abstract | 許多乳酸桿菌 (lactobacilli) 具有抵抗病原菌感染及免疫調節的功能,但其作用機制至今仍不清楚。本研究係將無特定病原 (specific pathogen free, SPF) 級 BALB/c 小鼠經由連續餵食本研究室自行分離菌株 108 colony forming units (CFU)/day 副乾酪乳桿菌副乾酪亞種 (Lactobacillus paracasei subsp. paracasei) NTU 101 菌液,藉此評估其免疫調節能力。
本論文首先利用連續餵食 L. paracasei subsp. paracasei NTU 101 不同時間後終止餵食,探討餵食乳酸桿菌不同的時間點對於先天性及後天性免疫調節能力的影響,以評估是否具有免疫調節的時間依賴性 (time-dependent)。結果顯示,連續餵食 3、6 及 9 週的 L. paracasei subsp. paracasei NTU 101 菌液後,具有增強小鼠脾臟中樹突狀細胞 (dendritic cell) 及自然殺手細胞 (natural killer cell) 活性、淋巴球增生及抗體分泌的能力,同時隨著餵食的時間越久,即使停止餵食乳酸桿菌一週後也能觀察到先天性及後天性免疫調節的活性上升,表示經由餵食 L. paracasei subsp. paracasei NTU 101 所引起的免疫調節反應具有時間依賴性。另外,本研究也觀察到餵食 L. paracasei subsp. paracasei NTU 101 後對於腸道中的益生菌 bifidobacteria 及 lactobacilli 數目有增加而有害菌 Clostridium perfringens 數目會減少,而此種腸道菌叢變化的情形可能與 L. paracasei subsp. paracasei NTU 101 的免疫調節時間依賴性能力有正相關性。 乳酸桿菌對全身性 (以脾臟代表) 及腸道局部性 (以腸道 Peyer’s patches 代表) 免疫中免疫細胞的作用機制仍不清楚,因此,本研究將連續餵食 3、6 及 9 週的 L. paracasei subsp. paracasei NTU 101 菌液進行各免疫組織中細胞活性的評估。結果顯示餵食 L. paracasei subsp. paracasei NTU 101 後,對於腸道 Peyer’s patches 中淋巴細胞數目增生及活性、腸道 IgA 抗體分泌、輔助型 T 細胞與樹突狀細胞間的交互作用及其細胞激素表現相較於脾臟細胞皆有較強的免疫調節反應,表示經由餵食 L. paracasei subsp. paracasei NTU 101 後對於不同的免疫組織會產生不同的免疫調節反應,而對於腸道 Peyer’s patches 有較強的免疫調節能力,可能也因此幫助局部性腸道黏膜免疫力 (gut mucosal immunity) 反應的發生。 由於 L. paracasei subsp. paracasei NTU 101 具有增強腸道免疫調節反應而可能具有抑制腸道病原菌感染的能力,因此,利用腸出血性大腸桿菌 (Enterohaemorrhagic, EHEC E. coli O157:H7) 感染小鼠前或後餵食 L. paracasei subsp. paracasei NTU 101 以評估乳酸桿菌是否能經由免疫調節而具有保護能力,結果顯示,感染前七天餵食 L. paracasei subsp. paracasei NTU 101 組對於樹突狀細胞的抗原呈現能力、輔助型 T 細胞的活性及抗體分泌相較於感染後才餵食乳酸桿菌的組別皆有較好的免疫調節能力。並且也觀察到在體重變化、發病率、阻斷 E. coli O157:H7 經由巨噬細胞上的 Toll-like receptors (TLRs) 接受器進行訊息傳遞的能力,以及在促發炎細胞激素與趨化激素的表現上,感染前給予乳酸桿菌組也有較佳的減緩能力。因此,本試驗證實 L. paracasei subsp. paracasei NTU 101 能經由免疫調節的能力減緩經由 E. coli O157:H7 感染所引起的發炎或發病能力,並且在感染前即投予乳酸桿菌相較於感染後才投予乳酸桿菌具有較強的保護能力。 本論文對於益生菌之調節免疫反應與食用時間的關連性及其作用機制進行探討,以幫助瞭解保健食品對於預防醫學的貢獻,第一次證實了乳酸桿菌的免疫調節反應為時間依賴性、不同的次級淋巴組織具有不同的免疫調控機制與對病原菌的保護能力,皆顯示了本實驗分離的乳酸桿菌菌株對人體可能具有益處,未來期望能繼續發展並深入探討乳酸桿菌的免疫調節機制及其他功效,期望日後能將本菌株加以實際運用至產業的規模,促進保健食品的蓬勃發展並且能夠為人類的健康帶來助益。 | zh_TW |
dc.description.abstract | Many Lactobacillus species regulate the immune responses, benefits in the protection of host organisms against a wide variety of enteropathogens, but the mechanisms of immunomodulation are still unknown. This study was investigated the immunomodulation in SPF BALB/c mice by diet with 108 colony forming units (CFU) of L. paracasei subsp. paracasei NTU 101.
The first section described the possible time-dependent role of L. paracasei subsp. paracasei NTU 101 in immunomodulation was investigated in BALB/c mice fed daily with 108 CFU of L. paracasei subsp. paracasei NTU 101 for 3, 6, and 9 weeks, and following feeding with Lactobacillus-free food for a further 7 days. We observed up-regulation of the antigen-presenting ability of dendritic cells, and expression of natural killer group-2 D (NKG2D) molecules capable of trigger natural killer cell-mediated cytotoxicity. Lymphocyte proliferation and antibody production were also significantly increased in mice after treatment. Innate and adaptive immunity remained constant even at the most protracted feeding time, indicative of the time dependence of the bacterial-mediated enhanced immunity. To better correlate intestinal microflora with immunity, the intestinal contents of probiotics and harmful microorganisms were determined. Results showed an altered intestinal microflora, with increases in bifidobacteria and lactobacilli and a decreased content of Clostridium perfringens after feeding with L. paracasei subsp. paracasei NTU 101. It is possible that persistent activation of immunity might be induced by intestinal probiotics. The role of lactic acid bacteria in gut mucosal immunity was investigated by comparing the enhanced effects in the Peyer’s patches and spleen of BALB/c mice fed daily with L. paracasei subsp. paracasei NTU 101 for 3 , 6, and 9 weeks. After feeding with Lactobacillus, Peyer’s patches exhibited a more distinct capacity to induce CD4+ T cell-dendritic cell interactions, lymphocyte proliferation, and cytokine secretion than the spleen, and thereby promoted greater intestinal IgA production that could enhance gut mucosal immunity. This study investigated the immunomodulation of Lactobacillus against enterohemorrhagic Escherichia coli O157:H7 infection in a BALB/c mice models. Mice were fed with L. paracasei subsp. paracasei NTU 101 for 7 days, before and after challenge with E. coli O157:H7. We observed up-regulation of the antigen-presenting ability of dendritic cells, helper T cell activation, and antibody production in post- and pre-treated mice, compared with untreated mice in the E. coli O157:H7 infection group. Moreover, Lactobacillus can increased post-challenge body weight gain and lower cumulative morbidity rates, down-regulate the expression of TLRs on macrophage, proinflammatory cytokines, and chemokines in the post- or pre-treated mice induced by E. coli O157:H7. We observed a greater enhancement in the immunity and inhibition of inflammation among the mice in the pre-treated group than in the post-treated group. These results demonstrated that feeding mice L. paracasei subsp. paracasei NTU 101 could reduce the severity of E. coli O157:H7 infection by modulating their immune response. The results also suggested that dietary supplementation with Lactobacillus strain could have a significant impact on immunological responses. The benefits were more significant when administered before rather than after infection. These studies, which represent the first report of the enhancement of immunity by Lactobacillus in time-dependent manner, exhibited a more distinct capacity of immunomodulation in secondary lymphoid tissue, and reduce the severity of E. coli O157:H7 infection by modulating their immune response. The results also suggested that dietary supplementation with Lactobacillus strain could have a significant impact on immunological responses. Therefore, application of L. paracasei subsp. paracasei NTU 101 may be an effective candidate for use as a probiotic in the prevention of gastrointestinal infection in humans, the more appropriate application worthy to be studied in the future. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:47:25Z (GMT). No. of bitstreams: 1 ntu-100-D94b47103-1.pdf: 7411003 bytes, checksum: 8a51e17be93f2ebef1367df3c850090d (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 第一章 文獻回顧………..………………………….…………………………..… 1
第一節 乳酸菌與益生菌之介紹………..…….…………………………..… 1 第二節 益生菌調節腸道菌相………..……………………………….…..… 2 第三節 益生菌激活免疫之主成份………..…………………….………..… 4 第四節 影響乳酸菌免疫調節的因素或機制………..……………….…..… 5 第五節 人體免疫系統簡介……………………..…….…………………..… 7 第六節 腸道免疫系統…………………………...………………………..… 12 第七節 腸出血性大腸桿菌介紹………………………….…………..…..… 14 第八節 研究目的………..……………….………………………………..… 20 第二章 乳酸桿菌對免疫調節的時間依賴性………..…………….…………..… 23 第一節 前言……………………….……………..………………………..… 23 第二節 材料方法………………..……….………………………………..… 23 1. 實驗菌株………………..……….……………………………….…..… 23 2. 乳酸桿菌之生長曲線…………..……………….….………………..… 24 3. 實驗動物及飼養環境…………..…………….……….……………..… 24 4. 調節免疫能力之動物試驗…………..…………………..…………..… 25 4.1. 劑量與分組…………..…….………………………………..…..… 25 4.2. 乳酸桿菌抗原製備…………..…….………………………..…..… 25 4.3. 蛋白質定量…………..……….……………………………..…..… 27 4.4. 脾臟中免疫細胞表面標記與活性之測定...…………...…...…..… 27 4.5. 血清收集…………..….……………………………………..…..… 32 4.6. 酵素免疫連結吸附法測定血清中總 IgG 抗體…………..…..… 32 5. 改善腸道菌相之動物試驗…………..….…………………….……..… 33 5.1. 劑量與分組…………..……….……………………………..…..… 33 5.2. 糞便樣本收集…………..…….……………………………..…..… 33 5.3. 菌種培養…………..…………………….…………………..…..… 34 5.4. 菌種確認…………..……………….………………………..…..… 35 6. 統計分析方法…………..…………….………………………….…..… 39 第三節 試驗結果…………..………….…………………………………..… 39 1. 餵食乳酸桿菌對免疫調節能力具有時間依賴性…………..……....… 39 1.1. L. paracasei subsp. paracasei NTU 101 對樹突狀細胞之抗原呈 現能力的表現具有時間依賴性……...……….……………..…..… 39 1.2. L. paracasei subsp. paracasei NTU 101 對自然殺手細胞之毒殺 能力具有時間依賴性………..……………………………….…..… 41 1.3. L. paracasei subsp. paracasei NTU 101 對淋巴球增生的能力具 有時間依賴性…………..…………….……….……………..…..… 46 1.4. 血清中總 IgG 抗體含量測定…………...………………...…..… 48 2. 乳酸桿菌具有改善腸道菌相的能力…………..…….……………..…. 48 2.1. 雙歧桿菌…………..……….………………………………..…..… 48 2.2. 乳酸桿菌…………..……….……………………………….…..…. 50 2.3. 產氣莢膜梭菌…………..…….……………………………..…..… 50 第四節 討論…………...…………………………………………………..… 52 第五節 總結…………...…………………………………………………..… 55 第三章 乳酸桿菌對不同淋巴組織之免疫調節影響…………...……………..… 56 第一節 前言…………….……………………………………………..…..… 56 第二節 材料方法…………...……………………………………………..… 56 1. 實驗菌株…………..….………………………………………….…..… 56 2. 劑量與分組…………..……….………………………………….…..… 56 3. 實驗動物及飼養環境…………..……….……………………….…..… 57 4. 脾臟與腸道 Peyer’s patch 中免疫細胞表面標記與活性之測定…… 57 4.1. 小鼠脾臟內免疫細胞之分離……………...………………..…..… 57 4.2. 小鼠腸道 Peyer’s patch 淋巴結免疫細胞之分離….…..……..… 57 4.3. 脾臟及腸道 Peyer’s patch 中之細胞表面標記與活性分析….… 59 4.4 脾臟及腸道 Peyer’s patch 中淋巴球增生能力分析….…...…..… 59 5. 血清收集…………...…………………………………………….…..… 61 6. 酵素免疫連結吸附法測定血清中 IgA 抗體……...…………..…..… 61 7. 組織切片之製作及免疫組織化學染色……...………………….…..… 63 8. 樹突狀細胞及輔助型 T 細胞之純化及雷射掃描共軛焦顯微鏡之 觀察…….……..………………………………………………….…..… 64 8.1. 細胞前處理…………..….…………………………………..…..… 64 8.2. 以 MACS 技術純化樹突狀細胞與輔助型 T 細胞….…..…..… 64 8.3. 利用雷射掃描共軛焦顯微鏡觀察樹突狀細胞與輔助型 T 細胞 之表現…………...………………………………………….…..… 66 9. 反轉錄-即時聚合酶鏈反應分析基因表現…………..….……...…..… 66 9.1. 總核醣核酸萃取…………..…….…………………..…………..… 66 9.2. 反轉錄 cDNA 合成………...……………………………...…..… 67 9.3. 即時聚合酶鏈反應 (real-time PCR) ………...…………….…..… 67 10.統計分析方法…………...……………………………………….…..… 69 第三節 試驗結果…………..………….…………………………………..… 69 1. 餵食乳酸桿菌不同時間對脾臟及腸道 Peyer’s patch 中細胞數目之 影響……………...………………………………………….…..……… 69 1.1. 乳酸桿菌可促進脾臟及腸道 Peyer’s patch 中輔助型 T 細胞 數目增加……………..…….…..………………………………….. 69 1.2. 乳酸桿菌可促進脾臟中毒殺型 T 細胞數目增加………..…..… 69 1.3. 乳酸桿菌可促進腸道 Peyer’s patch 中 B 細胞數目增加…..…. 71 2. 乳酸桿菌促進腸道 Peyer’s patch 中輔助型 T 細胞活性…….....…. 71 3. 乳酸桿菌促進腸道中 IgA 分泌細胞的表現……....………….…..… 73 4. 乳酸桿菌促進腸道 Peyer’s patch 中樹突狀細胞與輔助型 T 細胞 產生交互作用…………...…………...………..……………..……..….. 73 5. 乳酸桿菌誘導腸道 Peyer’s patch 中細胞增生及細胞激素分泌…… 76 第四節 討論……...………………………………………………………..… 79 第五節 總結…………..………….………………………………………..… 81 第四章 乳酸桿菌對腸出血性大腸桿菌誘發小鼠免疫調節反應之影響….....… 82 第一節 前言…………..……….…………………………………………..… 82 第二節 材料方法…………..…….………………………………………..… 82 1. 實驗菌株…………..……….…………………………………….…..… 82 2. 劑量與分組…………..………….……………………………….…..… 83 3. 實驗動物及飼養環境…………..……….………………….………..… 85 4. 脾臟中免疫細胞表面標記與活性之測定…………...………….…..… 85 4.1. 小鼠脾臟內免疫細胞之分離…………..….………………..…..… 85 4.2. 脾臟中樹突狀細胞及巨噬細胞之細胞表面標記分析….……..… 85 5. 酵素免疫連結吸附法測定血清中抗體含量………….…….…..…..… 87 6. 反轉錄聚合酶鏈反應分析基因表現…………...……………….…..… 87 6.1. 總核醣核酸萃取及反轉錄 cDNA 合成………...………...…..… 87 6.2. 聚合酶鏈反應 (PCR) ……...…………………………………..… 89 7. 統計分析方法…………...……………………………………….…..… 89 第三節 試驗結果…………..….…………………………………………..… 89 1. 乳酸桿菌減緩經 E. coli O157:H7 感染小鼠的體重下降及發病率... 89 2. 乳酸桿菌增強被 E. coli O157:H7 感染的小鼠脾臟中樹突狀細胞 的活性……..….………………………………………………………... 91 3. 乳酸桿菌增強被 E. coli O157:H7 感染的小鼠脾臟中輔助型 T 細 胞表面分子的活性……...…………....………………………….…..… 91 4. 乳酸桿菌減緩被 E. coli O157:H7 感染的小鼠脾臟中巨噬細胞表 面 TLR 分子的活性………......…………...…………………..…..… 94 5. 乳酸桿菌與 E. coli O157:H7 對小鼠脾臟中細胞激素 mRNA 表現 情形之影響…………...…………….…………………………..……… 97 6. 乳酸桿菌增強被 E. coli O157:H7 感染小鼠血清中抗體的分泌.….. 97 第四節 討論…………..……….…………………………………………..… 101 第五節 總結…………..………….………………………………………..… 104 第五章 總結論…………..……….……………………………………………..… 105 第六章 參考文獻…………..…….……………………………………………..… 107 期刊附錄…………..…….…………………………………………………..…..… 124 | |
dc.language.iso | zh-TW | |
dc.title | 以小鼠模式探討副乾酪乳桿菌副乾酪亞種 NTU 101 之免疫調節機制 | zh_TW |
dc.title | Mechanism of immunomodulation by Lactobacillus paracasei subsp. paracasei NTU 101 in mice model | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 蘇遠志,黃健雄,陳明汝,繆希椿,蔡英傑,廖啟成 | |
dc.subject.keyword | 乳酸桿菌,腸出血性大腸桿菌,先天性免疫,腸道黏膜免疫調節,培耶氏斑, | zh_TW |
dc.subject.keyword | Lactobacillus,enterohemorrhagic E. coli O157:H7,Innate immunity,gut mucosal immunity,Peyer’s patches, | en |
dc.relation.page | 124 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-06-07 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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