生命科學院: 生化科技學系指導教授: 林璧鳳呂學耘Lu, Hsueh-YunHsueh-YunLu2017-03-022018-07-062017-03-022018-07-062016http://ntur.lib.ntu.edu.tw//handle/246246/272233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。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.13151759 bytesapplication/pdf論文公開時間: 2021/8/24論文使用權限: 同意有償授權(權利金給回饋學校)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bitter melonregulatory T cellintestinal immune systeminflammatory bowel diseasemesenteric lymph nodesthesis10.6342/NTU201601903http://ntur.lib.ntu.edu.tw/bitstream/246246/272233/1/ntu-105-D99b22006-1.pdf