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研究生: 曾建銘
Tseng, Chien Ming
論文名稱: 液化澱粉芽孢桿菌胞外多醣提升腸道上皮細胞的保護性功能
Bacillus amyloliquefaciens exopolysaccharides promote the protective functions of intestinal epithelial cells
指導教授: 鄭雪玲
Cheng, Hsueh-Ling
學位類別: 碩士
Master
系所名稱: 農學院 - 生物科技系
Department of Biological Science and Technology
畢業學年度: 110
語文別: 中文
論文頁數: 88
中文關鍵詞: exopolysaccharidesBacillus amyloliqufaciensCaco-2細胞腸道免疫力抗菌胜肽barrier functionp38Nrf2/HO-1 pathway
外文關鍵詞: exopolysaccharides, Bacillus amyloliqufaciens, Caco-2 cells, intestinal immunity, antimicrobial peptides, barrier function, p38, Nrf2/HO-1 pathway
DOI URL: http://doi.org/10.6346/NPUST202200078
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  • 益生菌有健胃整腸、抑制腸道發炎,以及促進腸道健康的功能,但是機制仍不完全清楚。於前期研究中,液化澱粉芽孢桿菌(Bacillus amyloliquefaciens) amy-1分泌的胞外多醣 (exopolysaccharides;EPS)被發現有抗發炎的功能。於是本研究以人類腸道上皮細胞Caco-2為模型,分析amy-1 EPS提升腸道上皮細胞的保護性功能。方法是以脂多醣 (Lipopolysaccharides;LPS)作為正對照組,以EPS單獨或與LPS共同處理Caco-2細胞,分析是否促進腸道上皮細胞的保護性功能,並初步研究其分子機制。結果,EPS能夠促進Caco-2細胞分泌抗菌胜肽並促進Caco-2細胞的屏障功能(barrier function),後者經分析認為與EPS促進occludin及zonula occludens-1等tight junction形成蛋白的表現有關。這些結果暗示EPS能提升腸道上皮細胞的抗菌能力及避免外來物質藉由細胞間隙進入身體的能力。EPS促進inducible nitric oxide synthase (iNOS), cyclooxygenase-2及interleukin-8的表現,暗示EPS可提升腸道上皮細胞的免疫相關活性。EPS活化nuclear factor erythroid 2–related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) pathway,並且降低活性氧物種 (reactive oxygen species)生成量,暗示EPS可提升細胞的抗氧化能力,而這可能與EPS活化 mitogen activated protein kinases中的p38有關。但是當LPS存在時,EPS卻又抑制iNOS的表現,暗示能抑制LPS引起的發炎反應。綜合這些結果,認為EPS可以提升腸道上皮細胞的保護性功能。

    Probiotics are beneficial to the digestive tract, can reduce intestinal inflammation, and promote the health of intestines, but the underlying mechanism is still unclear. The exopolysaccharides (EPS) of Bacillus amyloliqufaciens amy-1 were found to have anti-inflammatory effects. In this study, the human intestinal epithelial cell line Caco-2 was used as a model to investigate whether EPS can promte the protective functions of intestines. Caco-2 cells were treated with EPS or with lipopolysaccharides (LPS; positive control). Consequently, EPS increased the secretion of antimicrobial peptides from Caco-2 cells, and enhanced the barrier function of the cell. The latter was found to be associated with EPS-promoted expression of tight junction-forming proteins occludin and zonula occludens-1. These results suggest that EPS can improve the antibacterial activity and the ability to prevent foreign substances from entering the body through the intercellular space of intestinal epithelial cells. EPS promoted the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and interleukin-8, suggesting that EPS can enhance the immune-related activity of intestinal epithelial cells. EPS activated the nuclear factor erythroid 2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) pathway and reduced the production of reactive oxygen species, suggesting that EPS can enhance the antioxidant capacity of the cells, which may be related to the activation of EPS induced-p38 activation. However, in the presence of LPS, EPS inhibited the expression of iNOS, implying that EPS can inhibit LPS-induced inflammation. Taken together, EPS promote the protective functions of intestinal epithelial cells.

    中文摘要……………………………………………………………………………………………………………I
    Abstract…………………………………………………………………………………………………………II
    謝誌……………………………………………………………………………………………………………………III
    目錄………………………………………………………………………………………………………………………IV
    圖表目錄……………………………………………………………………………………………………………VII
    第1章、前言………………………………………………………………………………………………………1
    1.1 研究背景……………………………………………………………………………………………………1
    1.2 研究目的與架構………………………………………………………………………………………3
    1.3 研究設計及原理說明……………………………………………………………………………3
    1.4 研究成果對未來的影響…………………………………………………………………………5
    第2章、文獻回顧…………………………………………………………………………………………………6
    2.1 液化澱粉芽孢桿菌……………………………………………………………………………………6
    2.2 微生物的胞外多醣……………………………………………………………………………………7
    2.3 腸道的保護性功能對維持身體健康的重要性………………………………8
    2.3.1抗菌胜肽……………………………………………………………………………………………………11
    2.3.2腸道重要趨化因子Interleukin-8 (IL-8)……………………………12
    2.3.3 Barrier function及tight junction…………………………………13
    2.4發炎及其分子機制………………………………………………………………………………………14
    2.4.1 IKK/ NF-κB pathway的促發炎角色…………………………………………16
    2.4.2 Mitogen-activated protein kinase (MAPKs)的促發炎及抗氧化角色……………………………………………………………………………………………………………………………17
    2.5 Nuclear factor erythroid 2-related factor 2/ heme oxygenase-1 pathway的調節…………………………………………………………………19
    2.5.1 Nrf2的抗氧化角色………………………………………………………………………………19
    2.5.2 Antioxidant enzymes: HO-1, NQO-1, GCLM……………………………………………………………………………………………………………………………20
    2.6 Caco-2細胞作為腸道上皮細胞之模型……………………………………………22
    第3章、材料與方法………………………………………………………………………………………………23
    3.1實驗材料……………………………………………………………………………………………………………23
    3.1.1哺乳類動物細胞株……………………………………………………………………………………23
    3.1.2 EPS的製備及純度分析…………………………………………………………………………23
    3.1.3 藥品試劑……………………………………………………………………………………………………24
    3.1.4 細胞培養基………………………………………………………………………………………………26
    3.1.5 細胞處理之緩衝液及試劑……………………………………………………………………26
    3.1.6 細胞蛋白質粗萃液之試劑……………………………………………………………………27
    3.1.7 蛋白質電泳與西方墨點法試劑……………………………………………………………27
    3.1.8 酵素連結免疫分析(ELISA)…………………………………………………………………29
    3.1.9 抗體…………………………………………………………………………………………………………………29
    3.1.10 儀器設備………………………………………………………………………………………………………30
    3.2 實驗方法…………………………………………………………………………………………………………………31
    3.2.1 Caco-2細胞冷凍活化…………………………………………………………………………………31
    3.2.2 Caco-2細胞繼代培養(Subculture)……………………………………………………31
    3.2.3 細胞毒性分析……………………………………………………………………………………………………32
    3.2.4 以Real-time PCR分析抗菌胜肽的表現………………………………………………32
    3.2.5分析Caco-2細胞barrier function………………………………………………………36
    3.2.6 以ELISA分析TNF-α、IL-6、IL-8、human β-defensin2之分泌 …………………………………………………………………………………………………………………………………………………36
    3.2.7 細胞內ROS分析…………………………………………………………………………………………………37
    3.2.8 細胞核蛋白萃取………………………………………………………………………………………………37
    3.2.9 細胞蛋白質粗萃…………………………………………………………………………………………………38
    3.2.1蛋白質定量………………………………………………………………………………………………………………38
    3.2.11聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresie;SDS-PAGE)………………………………39
    3.2.12蛋白質轉漬及抗體分析………………………………………………………………………………………40
    3.2.13實驗數據統計分析…………………………………………………………………………………………………41
    第4章、結果……………………………………………………………………………………………………………………………42
    4.1 分析EPS及LPS對Caco-2細胞的毒性…………………………………………………………………42
    4.2 分析EPS是否促進抗菌胜肽表現…………………………………………………………………………42
    4.3 分析是否促進barrier function……………………………………………………………………43
    4.4 分析EPS是否提升免疫力……………………………………………………………………………………………43
    4.5 分析EPS是否活化Nrf2/HO-1 pathway……………………………………………………………44
    4.6 分析EPS是否引起Caco-2細胞發炎或減緩發炎………………………………………………45
    第5章、討論………………………………………………………………………………………………………………………………61
    第6章、結論………………………………………………………………………………………………………………………………70
    參考文獻………………………………………………………………………………………………………………………………………71

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