山苦瓜影響調節型T細胞進而調節腸道免疫之研究

Hsueh-Yun Lu; 呂學耘

請用此 Handle URI 來引用此文件： http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50086

完整後設資料紀錄

DC 欄位	值	語言
dc.contributor.advisor	林璧鳳
dc.contributor.author	Hsueh-Yun Lu	en
dc.contributor.author	呂學耘	zh_TW
dc.date.accessioned	2021-06-15T12:29:23Z	-
dc.date.available	2021-08-24
dc.date.copyright	2016-08-24
dc.date.issued	2016
dc.date.submitted	2016-08-05
dc.identifier.uri	http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50086	-
dc.description.abstract	慢性發炎和免疫失調與許多疾病的發生相關，包括胰島素抗性、過敏與發炎性大腸疾病等，調節型T細胞 (Regulatory T cells, Treg)具有抗發炎與調節免疫的作用。山苦瓜 (wild bitter melon, WBM) 已被應用於改善胰島素抗性，但免疫相關的研究仍少，因此本研究探討山苦瓜對腸道免疫系統及調節型T細胞的影響。以高油飲食誘導肥胖的C57BL/6J小鼠，探討山苦瓜是否透過抗發炎與免疫調節改善胰島素抗性，結果餵食含5％山苦瓜凍乾粉末飼料12週，降低小鼠禁食血糖和血脂，改善胰島素抗性，降低脂肪組織SVF (stromal vascular fraction) M1/M2的比例與CD4+ T細胞的數量。同時，山苦瓜增加腸繫膜淋巴結 (mesenteric lymph nodes, MLN) 的CD4+CD25+Foxp3+ Treg比率與數量。以餵食Foxp3-EGFP小鼠山苦瓜驗證山苦瓜對腸道免疫及Treg的影響，結果顯示山苦瓜顯著增加Foxp3-EGFP小鼠MLN的CD4+CD25+Foxp3+ Treg比率與數量，且MLN與皮耶氏體細胞分泌較高的IL-10及TGF-β，再次顯示山苦瓜具有促進腸道Treg免疫之作用。為探討山苦瓜各萃物對增加調節型T細胞的分化潛力與機制，以山苦瓜各萃物添加於初代脾臟、MLN、CD4+ T與樹突細胞培養試驗培養液中。結果山苦瓜M-Res與BuE萃物可促進初代脾臟細胞IL-10和TGF-β分泌，透過aryl hydrocarbon receptor (AhR) 增加CD4+ T細胞分化為Foxp3+ Treg。而WE萃物促進初代樹突細胞IL-10分泌，且WE萃物培養後的樹突細胞可增加CD4+ T細胞分化為 IL-10+ T細胞與Foxp3+ Treg。實驗續以BuE萃物純化分離並鑑定活性成份，結果發現增加CD4+ T細胞分化為Foxp3+ Treg成份之一為tryptophan。最後，以dextran sulfate sodium (DSS) 誘發腸炎小鼠模式，驗證山苦瓜與tryptophan是否透過增加Treg，減緩腸道發炎反應。實驗於DSS誘發腸炎前餵食小鼠含5%山苦瓜凍乾粉飼料或0.5% tryptophan飼料共2週或6週，結果山苦瓜能顯著增加MLN內Foxp3+ Treg及大腸Foxp3 mRNA基因表現量，並降低大腸組織IL-6與IL-17，增加IL-10含量，減緩DSS誘發小鼠體重流失、疾病指數與腸道縮短現象。2週補充tryptophan顯著降低大腸組織IL-6含量，減緩小鼠疾病指數。大腸組織TNF-α、IL-6、IL-1β、IL-17含量以及疾病指數，皆與Foxp3 mRNA表現量呈現顯著負相關，顯示山苦瓜透過增加腸道中Treg，降低腸道發炎反應，減緩DSS誘發腸炎小鼠疾病指數。綜合上述結果，山苦瓜具有促進小鼠腸道調節型T細胞免疫作用，減緩DSS誘發腸炎小鼠腸道發炎。山苦瓜促進調節型T細胞免疫的活性萃物包含M-Res、BuE和WE萃物，可能的機制為活化AhR，促進Foxp3的表現；增加CD103+ 樹突細胞比率及IL-10分泌，促進Foxp3+ Treg與IL-10+CD4+ T細胞分化。顯示山苦瓜具有抗發炎與免疫調節之功效，維持腸道免疫平衡，可應用於預防及減緩發炎性腸道疾病，做為腸道保健之蔬果，與開發為腸道保健相關食品的潛力。	zh_TW
dc.description.abstract	Chronic inflammation and immune disorder are associated with insulin resistance, allergy, inflammatory bowel disease, etc. Regulatory T lymphocytes (Treg) suppress a variety of inflammatory and pathological immune responses. Wild bitter melon (WBM) has been demonstrated to improve the insulin resistance, but its effect on immune is still unclear. Therefore, the aim of this study is to investigate the effect of WBM on the intestinal immune system and Treg development. First, to investigate the improvement in insulin resistance of wild bitter melon is involved with its immune modulatory effects. High-fat diet-induced obese C57BL/6J mice were fed the diet containing 5% WBM powder for 12 weeks. The results showed that WBM treatment reduced fasting glucose and triglyceride in the serum, and improved insulin resistance. WBM reduced the M1/M2 ratio and CD4+ T cells infiltration in stromal vascular fraction (SVF). Moreover, WBM increased the percentage of CD4+Foxp3+ Treg in mesenteric lymph nodes (MLN). To confirm the effects of WBM on intestinal immune system and Foxp3+ Treg cells population, Foxp3-EGFP mice co-expressing EGFP and Foxp3 in Treg were fed WBM experimental diet. The results showed that WBM also increased the percentage of CD4+ Foxp3+ Treg in MLN and interleukin (IL)-10 and TGF (transforming growth factor)-β secretion by ConA-stimulated Peyer’s patches and MLN cells. These results suggested that WBM could enhance the Foxp3+ Treg development in the intestinal immune system. The bioactive compounds of WBM and mechanisms of promoting the development of Treg were investigated by using the primary spleen cells, MLN cells, CD4+ T cells and bone marrow derived dendritic cells (BMDC) culture in vitro. The results showed that M-Res and BuE extract of WBM increased IL-10 and TGF-β secretion by splenocytes and promoted the development of Foxp3+ Treg through the aryl hydrocarbon receptor (AhR) pathway. WE extract increased IL-10 secretion by BMDC and promoted CD4+ T cells differentiate into IL-10+ T cells and Foxp3+ T cells in CD4+ T cells cocultured with WE pretreated BMDC. Moreover, tryptophan is the bioactive compound from BuE extract was found that increase Foxp3+ Treg development in vitro. The anti-inflammatory and immunomodulatory effects of WBM and tryptophan on inflammatory bowel disease were investigated by using dextran sodium sulphate (DSS)- induced colitis murine model. A diet containing 5% WBM or 0.5% tryptophan was administered to mice for 2 or 6 weeks and then gave 7 days DSS treatment. WBM intake significantly improved weight loss, disease activity index and colon shortening. Colonic cytokine IL-6 and IL-1β were significantly decreased, and the anti-inflammatory cytokine IL-10 was increased in WBM-fed mice. The mRNA expression of TGF-β and Foxp3 in colon, and Foxp3+ Treg cell population in the MLN were also significantly higher in the WBM group. Tryptophan supplement ameliorated disease activity index and reduced the colonic IL-6 levels. The significantly negative correlation between Foxp3 mRNA expression and pro-inflammatory cytokines in colon is in accordance with the critical role of Treg cell for regulation of intestinal immune homeostasis. These results suggested that WBM reduces the inflammatory response and induces higher level of Treg cells in DSS-induced colitic mice. In summary, WBM enhanced Treg development in intestinal immune system in both in vitro and in vivo. The mechanism of enhancing Treg development by WBM are not only through AhR pathway, but also increased the CD103+ dendritic cells and IL-10 secretion. Furthermore, WBM inhibited the inflammatory response caused by colitis and induces higher level of Treg cells in the intestinal immune system. These results suggested that WBM could exert the anti-inflammatory and immunoregulatory effects in maintaining intestinal immune balance and alleviating the severity of the symptoms on inflammatory bowel disease.	en
dc.description.provenance	Made available in DSpace on 2021-06-15T12:29:23Z (GMT). No. of bitstreams: 1 ntu-105-D99b22006-1.pdf: 13151759 bytes, checksum: ed1b28a22ca0ed123305a16331ec6fe4 (MD5) Previous issue date: 2016	en
dc.description.tableofcontents	摘要......................................................................................................... Ⅰ 目錄........................................................................................................ V 表目錄................................................................................................... X 圖目錄.................................................................................................... XI 縮寫對照表.............................................................................................. XIV 第一章、序論第一節文獻回顧一、苦瓜 .............................................................................................1 （一）苦瓜之介紹 .............................................................................. 1 （二）苦瓜之相關研究...........................................................................2 二、腸道免疫系統................................................................................7 三、調節型T細胞..................................................................................8 （一）調節型T細胞的介紹......................................................................8 （二）調節型T細胞抑制作用的機制 ..................................................... 10 （三）營養素與食材對於調節型T細胞的影響...........................................11 三、肥胖與胰島素抗性..........................................................................15 （一）胰島素抗性 ..............................................................................16 （二）胰島素抗性與發炎反應 ............................................................. 17 （三）調節型T細胞與胰島素抗性 ..................................................... 19 （四）其他免疫細胞與胰島素抗性 ..................................................... 20 四、發炎性腸道疾病............................................................................ 20 （一）發炎性腸道疾病的介紹 ............................................................. 20 （二）發炎性腸道疾病與免疫 ..............................................................21 （三）調節型T細胞與發炎性腸道疾病 ..............................................22 （四）小鼠腸炎模式.............................................................................23 第二節研究動機與目的...........................................................................26 第二章、山苦瓜對高油飲食誘導肥胖小鼠免疫反應的影響第一節前言...........................................................................................28 第二節材料與方法一、動物飼養.......................................................................................29 二、血清相關指標分析..........................................................................31 三、組織細胞分離與培養 ......................................................................33 四、細胞激素測定................................................................................35 五、免疫細胞表型分析.........................................................................36 六、脂肪組織基因表現量分析...............................................................38 七、統計方法 ......................................................................................40 第三節結果一、山苦瓜對高油飲食誘導肥胖小鼠生長情形及攝食量的影響..................41 二、山苦瓜對高油飲食誘導肥胖小鼠禁食血糖及血脂的影響.....................41 三、山苦瓜對高油飲食誘導肥胖小鼠口服葡萄糖耐受性測試的影響...........42 四、山苦瓜對高油飲食誘導肥胖小鼠血清指標與組織重的影響..................43 五、山苦瓜對高油飲食誘導肥胖小鼠脂肪組織SVF免疫細胞表型分析的影響 45 六、山苦瓜對高油飲食誘導肥胖小鼠脂肪組織mRNA基因表現量的影響.....46 七、山苦瓜對高油飲食誘導肥胖小鼠MLN及脾臟內CD4+T細胞與Foxp3+T細胞的比率的影響..............................................................................47 八、山苦瓜對高油飲食誘導肥胖小鼠脾臟及MLN細胞的分泌細胞激素的影響48 第四節討論一、山苦瓜減緩高油飲食誘導肥胖小鼠的胰島素抗性與脂肪組織發炎.........50 二、山苦瓜改善高油飲食誘導肥胖小鼠胰島素抗性與脂肪發炎可能機制......52 三、山苦瓜對高油飲食小鼠腸道免疫系統的影響......................................53 第五節結論..........................................................................................54 第三章、山苦瓜對Foxp3-EGFP小鼠腸道免疫與調節型T細胞的影響第一節前言...........................................................................................55 第二節材料與方法一、動物飼養.....................................................................................56 二、組織細胞分離與培養......................................................................57 三、細胞激素測定與免疫細胞表型分析...................................................57 四、Violet proliferation assay..............................................................57 五、統計方法.......................................................................................59 第三節結果一、山苦瓜對Foxp3-GFP小鼠生長情形與攝食的影響..............................60 二、山苦瓜對Foxp3-GFP小鼠淋巴組織細胞數、CD4+ T細胞、調節型T細胞與B 細胞比率......................................................................................61 三、山苦瓜對Foxp3-GFP小鼠淋巴組織細胞分泌細胞激素的影響..............65 第四節討論............................................................................................67 第五節結論............................................................................................69 第四章、篩選山苦瓜促進Treg分化之有效萃物與機制探討第一節前言 .......................................................................................70 第二節材料與方法一、實驗設計 .......................................................................................71 二、山苦瓜萃取物之製備........................................................................72 三、初代脾臟、MLN與樹突細胞培養.......................................................73 四、CD4+ T細胞誘導分化調節型T細胞....................................................75 五、CFSE proliferation assay...............................................................75 六、動物實驗設計與方法 .......................................................................76 七、統計方法........................................................................................79 第三節結果一、山苦瓜萃物促進小鼠初代脾臟與MLN細胞IL-10和TGF-β分泌...............80 二、山苦瓜M-Res及BuE萃物增加CD4+ T細胞分化為Treg.........................83 三、山苦瓜M-Res與BuE萃物對Treg表面分子和抑制T細胞增生的影響 ........85 四、M-Res與BuE萃物經由aryl hydrocarbon receptor (AhR) 促進Treg分化87 五、山苦瓜萃物對小鼠骨髓衍生樹突細胞的影響.........................................87 六、山苦瓜WE萃物對樹突細胞與T細胞共培養的影響.................................89 七、山苦瓜及其萃物對Foxp3-EGFP小鼠生長及攝食量與臟器重的影響.......91 八、山苦瓜及其萃物對Foxp3-EGFP小鼠Treg、IL-10+CD4+、IL-17+CD4+ 細胞與CD103+ 樹突細胞的影響..........................................................92 九、山苦瓜及其萃物對Foxp3-EGFP小鼠PP與MLN細胞mRNA基因表現量的影響.................................................................................................96 十、山苦瓜及其萃物對Foxp3-EGFP小鼠MLN與脾臟細胞激素分泌的影響...97 第四節討論一、山苦瓜各萃物對初代脾臟與MLN細胞的細胞激素分泌影響...................98 二、山苦瓜M-Res與BuE萃物為增加小鼠Treg有效萃物.............................99 三、山苦瓜WE萃物透過對樹突細胞的影響增加IL-10+ CD4+與Foxp3+Treg100 四、初代細胞培養與動物實驗之一致性.....................................................101 第五節結論.............................................................................................103 第五章山苦瓜增加調節型T細胞活性成份之純化分離第一節前言............................................................................................104 第二節材料與方法一、BuE萃取物之開放式逆向管柱層析.....................................................104 二、製備級高效率液相色層分析...............................................................105 三、統計分析 ........................................................................................105 第三節結果一、山苦瓜BuE萃物區分純化、結構鑑定..................................................106 二、色胺酸和腺苷對調節型T細胞分化的影響.............................................112 第四節討論一、色胺酸對調節型T細胞的影響.............................................................113 二、腺苷對調節型T細胞的影響................................................................114 第五節結論.............................................................................................114 第六章山苦瓜與色胺酸對DSS誘發腸炎小鼠發炎與免疫的影響第一節前言.............................................................................................115 第二節材料與方法一、實驗設計 ........................................................................................116 二、動物飼養.........................................................................................116 三、DSS誘發小鼠腸炎............................................................................117 四、大腸相關指標分析.............................................................................118 五、血清細胞激素含量分析.......................................................................119 六、免疫細胞比例與MLN細胞培養與細胞激素分析......................................119 七、統計分析 .........................................................................................119 第三節結果一、山苦瓜與色胺酸對腸炎小鼠體重流失與疾病指標分數的影響...................120 二、山苦瓜與色胺酸對腸炎小鼠各組織重的影響..........................................122 三、山苦瓜與色胺酸對腸炎小鼠大腸長度與組織型態的影響..........................122 四、山苦瓜與色胺酸對腸炎小鼠大腸組織發炎與免疫的影響..........................125 五、山苦瓜與色胺酸對腸炎小鼠血清IL-6含量的影響....................................126 六、山苦瓜與色胺酸對腸炎小鼠Treg與CD103+DC比例的影響......................127 七、山苦瓜與色胺酸對腸炎小鼠MLN細胞激素分泌的影響.............................128 八、山苦瓜與色胺酸對腸炎小鼠血清與腸組織serotonin含量的影響...............129 第四節討論一、DSS誘發BALB/c小鼠腸炎模式..........................................................130 二、山苦瓜減緩DSS誘發腸炎小鼠腸道發炎與症狀......................................131 三、色胺酸對DSS誘發腸炎小鼠腸道發炎的影響.........................................133 第五節結論..............................................................................................135 第七章綜合討論與結論................................................................................136 參考文獻....................................................................................................145
dc.language.iso	zh-TW
dc.subject	苦瓜	zh_TW
dc.subject	調節型T細胞	zh_TW
dc.subject	苦瓜	zh_TW
dc.subject	腸繫膜淋巴結	zh_TW
dc.subject	發炎性腸道疾病	zh_TW
dc.subject	腸繫膜淋巴結	zh_TW
dc.subject	發炎性腸道疾病	zh_TW
dc.subject	腸道免疫	zh_TW
dc.subject	調節型T細胞	zh_TW
dc.subject	腸道免疫	zh_TW
dc.subject	bitter melon	en
dc.subject	mesenteric lymph nodes	en
dc.subject	inflammatory bowel disease	en
dc.subject	intestinal immune system	en
dc.subject	regulatory T cell	en
dc.subject	bitter melon	en
dc.subject	mesenteric lymph nodes	en
dc.subject	inflammatory bowel disease	en
dc.subject	intestinal immune system	en
dc.subject	regulatory T cell	en
dc.title	山苦瓜影響調節型T細胞進而調節腸道免疫之研究	zh_TW
dc.title	Study on the effect of wild bitter melon on regulatory T cells and further modulation of intestinal immune system	en
dc.type	Thesis
dc.date.schoolyear	104-2
dc.description.degree	博士
dc.contributor.oralexamcommittee	江伯倫,江孟燦,林金源,謝佳倩,莊雅惠
dc.subject.keyword	苦瓜,調節型T細胞,腸道免疫,發炎性腸道疾病,腸繫膜淋巴結,	zh_TW
dc.subject.keyword	bitter melon,regulatory T cell,intestinal immune system,inflammatory bowel disease,mesenteric lymph nodes,	en
dc.relation.page	167
dc.identifier.doi	10.6342/NTU201601903
dc.rights.note	有償授權
dc.date.accepted	2016-08-07
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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