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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳明汝 | |
dc.contributor.author | Vivian Pang Yee Jia | en |
dc.contributor.author | 彭億佳 | zh_TW |
dc.date.accessioned | 2021-07-11T14:43:32Z | - |
dc.date.available | 2026-08-11 | |
dc.date.copyright | 2016-10-14 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-11 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78140 | - |
dc.description.abstract | 本實驗室先前自不同的克弗爾粒中分離出兩株具有潛力的益生菌,分別是Lactobacillus kefiranofaciens M1以及L. mali APS1。此兩株乳桿菌在體外以及體內試驗中皆具有抗腸炎的功效以外, L. kefiranofaciens M1也被證實具有維持腸道恆定、抗過敏及抗氣喘等免疫調節功效。然而,此二株菌的免疫調節機制尚未完全瞭解。在腸道上皮屏障內存在著一些稱為樹突細胞(dendritic cell, DC)的專職抗原呈現細胞,它們作為先天免疫與後天免疫反應的重要連結,因此扮演著影響輔助型T細胞分化的重要角色。因此本試驗希望可藉由樹突細胞瞭解此二株乳桿菌之免疫調節機制。
試驗將分離自C57BL/6小鼠骨髓的樹突細胞與不同濃度的L. kefiranofaciens M1及L. mali APS1進行共培養,以了解此二株乳桿菌對樹突細胞成熟和功能上的影響。結果顯示活菌L. mali APS1可刺激樹突細胞分泌與活化相關的細胞激素如tumor necrosis factor(TNF)-a、Interleukin(IL)-6及T helper(Th)1相關細胞激素IL-12,且當菌數增加後這些細胞激素皆有上升的現象,顯示L. mali APS1對樹突細胞的活化是具有正向劑量效應的。L. kefiranofaciens M1組別也有相似的結果,然而107 cells/mL L. kefiranofaciens M1組別刺激樹突細胞的IL-12分泌量較106 cells/mL L. kefiranofaciens M1組別少,這可能是因為IL-10大量的增加而抑制過多IL-12的分泌。比起活菌,此二株乳桿菌在熱致死後較不刺激樹突細胞分泌細胞激素。相反地,無論是活菌抑或熱致死菌,它們皆可刺激樹突細胞上調表面的major histocompatibility complex(MHC) class II及共刺激分子CD40、CD80、和CD86的表現量。除了108 cells/mL活菌 L. kefiranofaciens M1(樹突細胞存活率為30%)外,試驗中並無觀察到其他組別對樹突細胞產生細胞毒性。 進一步探討此二株乳桿菌誘導樹突細胞活化的訊號傳遞路徑,包括mitogen-activated protein kinases(MAPK)與 nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)路徑。當樹突細胞經過JNK抑制劑處理後,此二株乳桿菌可誘導樹突細胞上調CD40、CD86以及MHC class II的程度下降了。然而,在三種MAPK抑制劑的作用下,TNF-a與IL-10產量卻都降低了。NF-κB 的抑制也下調了樹突細胞表面的CD40表現量。因此,MAPK與NF-κB 路徑皆可能參與此二株乳桿菌影響樹突細胞生理功能的調控。為了了解此二株乳桿菌在體內是否亦會刺激樹突細胞活化,試驗以管餵方式給予健康BALB/c小鼠連續28天的活菌L. kefiranofaciens M1和L. mali APS1,並分析在培氏結、腸繫膜淋巴結與脾臟內調節型樹突細胞(CD103+ DC)、活化的樹突細胞(CD80+ DC)和調節型T細胞的數量。然而在所有組別中皆無顯著差異。由本試驗得知L. kefiranofaciens M1和L. mali APS1可活化樹突細胞的成熟與功能,然詳細機制仍需要更進一步的探討。 | zh_TW |
dc.description.abstract | Lactobacillus kefiranofaciens M1 and L. mali APS1, previously isolated from different kefir grains in our lab, are potential probiotic strains. Both strains demonstrated an anti-colitis effect in vitro and in vivo. L. kefiranofaciens M1 was also proven to possess immunoregulatory ability on sustaining intestinal homeostasis, anti-allergic and anti-asthmatic effects. However, the mechanisms of their immunomodulatory effect are still remain unclear. Dendritic cells (DCs), existing in the epithelial barrier, are professional antigen-presenting cells which play a pivotal role in connection of innate and adaptive immune systems, and then shaping helper T cells. Thus, the objective of this study was to investigate the immunomodulatory effects of our probiotic strains through DCs.
In this study, we isolated bone marrow derived-DCs (BMDCs) from C57BL/6 mice and co-cultured with various concentrations of L. kefiranofaciens M1 or L. mali APS1 to evaluate the effects of these two strains on DC maturation and functions. Results revealed that live L. mali APS1 could activate the production of DC maturation-related cytokines which are tumor necrosis factor (TNF)-a, Interleukin (IL)-6, and IL-12 in the resultant culture medium. By increasing the concentration of L. mali APS1 to 108 cells/mL, the production of cytokines further increased when compared to 107 cells/mL L. mali APS1 treatment group, suggesting the maturation of DC could be activated by L. mali APS1 in a dose-dependent manner. Similar results were shown in L. kefiranofaciens M1, however 107 cells/mL of M1 treatment group had slightly lower IL-12 production than in 106 CFU/mL of M1 treatment group, the suppression of IL-12 levels in high concentration M1 group maybe attribute to the elevated production of IL-10 in this group. Heat-killed bacteria showed little or no effect on cytokine production when compared to their live bacteria. Conversely, both of live and dead bacteria of these two strains could stimulate maturation of DCs by up-regulating surface expression of CD40, CD80, CD86 which are co-stimulatory molecules of DC maturation, as well as major histocompatibility complex (MHC) class II. No cytotoxicity effect was observed except for 108 cells/mL of live L. kefiranofaciens M1 treatment group (the cell viability of DCs was 30%). Furthermore, we investigated the signal transduction pathways engaged by these two strains, including mitogen-activated protein kinases (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway using specific inhibitors of p38 MAPK, JNK, MEK1/2 and NF- | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:43:32Z (GMT). No. of bitstreams: 1 ntu-105-R03626002-1.pdf: 7617614 bytes, checksum: 131ded6b0254397c28fe7673b563e6d2 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 誌謝………………………….…………………………………….……………………i
序言………………………….…………………………………….……………………iii 中文摘要……………………………………………………………………………...iv 英文摘要………………………………………………………………………………vi 壹、 文獻探討……………………………………………………………………...….1 第一節:本研究室自克弗爾(kefir)分離出的乳桿菌株………………...…1 一、 Lactobacillus kefiranofaciens M1………………………….………...1 二、 Lactobacillus mali APS1…………………………………………….1 第二節:益生菌與免疫調節…………………………………………………....4 第三節:樹突細胞…………………………………………………..….………...6 一、 樹突細胞活化之共刺激分子………………………….……………10 二、 樹突細胞活化之細胞激素…………………………….……………10 第四節:益生菌與樹突細胞的關係………………………………………….12 一、 體外試驗………………………………………………..………….12 (一)益生菌活菌……………………………………………………12 (二)益生菌死菌……………………………………………………12 (三)益生菌其他有效成分………………………………………….13 二、 體內試驗…………………….………………………………..……14 三、益生菌活化樹突細胞之可能訊息傳遞路徑………………..….15 試驗目的………………..…………………………..…………………………..21 貳、 材料與方法………………..…………………………..…………………………22 第一節:藉由體外試驗測試Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1可否刺激樹突細胞成熟………………..…………………22 一、 試驗菌株之活化與保存………………………..…………………22 (一) 試驗菌株…………………………..……………………..…….22 (二) 菌株之活化………………….………..…………………..……22 (三) 菌株之保存…………………...………………..………………22 二、 試驗細胞與製備方法…..…………………………..………………23 (一) 細胞培養液配製………………………..……………..………23 (二) 樹突細胞之製備…..…………………………..………….….23 三、 菌株與樹突細胞之共培養…..…………………………..………..24 (一) 菌株之製備…..…………………………..………………….24 (二) 菌株與細胞之共培養…..…………………………..…………24 四、 細胞存活率試驗………………………..…………………………25 五、 樹突細胞成熟之測定…………………..…………………………25 六、 細胞激素測定………………………..……………………………26 七、 統計分析………………………..…………………………………26 第二節:以雷射掃描共軛焦顯微鏡觀察樹突細胞與Lactobacillus mali APS1之相互作用……………………………………………….…...………27 一、試驗菌株之活化………………………………………………..……27 二、樹突細胞之製備………………………………………………..……27 三、細胞樣品之製備………………………………………………..……27 第三節:藉由體外試驗探討Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1活化樹突細胞之可能路徑………...……………...………28 一、 試驗菌株之活化………………………..…………………………28 (一) 試驗菌株………………………..……………………..………28 (二) 菌株之活化………………………..……………………...28 二、 樹突細胞的製備………………………..……………………...……28 三、 蛋白抑制試驗………………………..………………..……….……28 (一)MAPK路徑………………………..………....………..………29 (二)NF-kB路徑………………………..………....………..………29 四、樹突細胞成熟之測定………………………..………....…………29 五、細胞激素測定………………………..………..............……………30 六、統計分析………………………..………................…………………30 第四節:以動物試驗探討Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1在體內對樹突細胞與調節型T細胞的影響……………...31 一、 試驗菌株………………………..………................……………31 二、 實驗動物………………………..………................……………31 (一) 動物來源與飼養………..………...................………………31 (二) 試驗設計………..………................………...………………31 (三) 淋巴組織細胞分離………..………...................……………33 (四) 細胞染色分析………..………................……...……………33 參、 結果與討論………………………..………................…………………………35 第一節:以體外共培養試驗Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1對樹突細胞成熟之影響…………………….…………….35 一、 Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1對樹突細胞分泌細胞激素之影響………………………………….…..35 (一) 促發炎反應激素……………………………………………..36 (二) Th1相關細胞激素……………………………….…………..40 (三) 抗發炎反應細胞激素……………………………….………43 二、 Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1對樹突細胞表面分子表現量之影響………………………….……….…45 (一) MHC class II……………….……………….…………….….45 (二) 共刺激分子……………….…..…………….…………….…47 三、 Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1對樹突細胞存活率之影響…..…………….……………………………….51 第二節:以共軛焦顯微鏡觀察樹突細胞與Lactobacillus mali APS1的相互作用….…………………………………………………………..….….54 第三節:Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1刺激樹突細胞活化之訊息傳導路徑探討….………………………………...56 一、 NF-B路徑……………..………...............…………………………56 二、 MAPK路徑……………..………...............…………………………59 (一) 樹突細胞於MAPK不同路徑抑制後表面分子表現量之影響………..………...............………………………………….59 (二) 樹突細胞於MAPK不同路徑抑制後細胞激素分泌量之影響………..………...............………………………………….60 第四節:藉由動物試驗探討Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1對樹突細胞的活化…………………………………..……68 一、 Lactobacillus kefiranofaciens M1及Lactobacillus mali APS1影響淋巴器官中DC與Treg細胞的分佈情形...………...............................68 肆、 結論...………..............................……………………………………………….74 參考文獻...………..............................………………………………….…………….75 | |
dc.language.iso | zh-TW | |
dc.title | 藉由樹突細胞探討克弗爾分離乳桿菌免疫調節之機制 | zh_TW |
dc.title | Investigating the Mechanisms of Immunomodulatory Effects of Kefir Lactobacilli through Dendritic Cell | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 陳彥伯 | |
dc.contributor.oralexamcommittee | 潘子明,洪偉盛,何尚哲 | |
dc.subject.keyword | 樹突細胞,乳桿菌,免疫調節,NF-κB,MAPK, | zh_TW |
dc.subject.keyword | dendritic cell,lactobacilli,immunomodulation,NF-κB,MAPK, | en |
dc.relation.page | 90 | |
dc.identifier.doi | 10.6342/NTU201602355 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-11 | |
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 |
dc.date.embargo-lift | 2026-08-11 | - |
顯示於系所單位: | 動物科學技術學系 |
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