臺灣博碩士論文加值系統

腸道黏膜免疫系統是個很特別的地方，一方面能夠避免腸內共生菌所引起的免疫反應，一方面又能抵禦外來病原菌的侵略，兩種截然不同的免疫反應，在正常人的體內能達到平衡的狀態。近年來，許多文獻指出，腸道上皮細胞在調節腸道免疫系統上扮演重要的角色，例如利用CX3CL這類chemokine使dendritic cell趨向Th2的免疫走向，或是抑制macrophage 在lamina propria 中表現某些特定的receptor，避免對commensal過度反應而引發慢性發炎的疾病。有鑑於腸道上皮細胞的重要性，本論文旨在建立可以評估中草藥多醣在調節細胞和活體動物的免疫功能的平台，並希望能進一步探討中草藥多醣調控腸道免疫的機制之所在。
在in vitro IEC-6實驗中，可以看到靈芝多醣、北耆多醣對於IL-6、TNF-α、TLR(Toll-like receptor)7、TLR9的基因表現有促進的作用，可能具有具有提升innate immunity的能力，能夠幫助免疫細胞的活化；另一方面，石斛多醣能夠大幅提昇TGF-β汹汹的基因表現，顯示石斛多醣可能具有誘導免疫抑制的路徑。
在in vivo 實驗中，我們利用OVA 引起的氣喘小鼠測試石斛多醣的治療效果，結果發現石斛多醣在30和90 mg/kg/day的劑量給予下，能夠顯著性的降低呼吸道阻力，達到氣喘減緩的目的。同時在肺部沖洗液中，能夠顯著性的減少免疫細胞和嗜酸性白血球（eosinophil）的浸潤現象，在肺部切片上也觀察到肺部的發炎情況有被抑制。除此之外，我們純化Lamina propria和肺部淋巴節（mediastinal lymph node, MLN）的細胞，進行CD4+CD25+的細胞染色，發現給予石斛多醣的小鼠，在腸道中的此類regulatory T cells的比例有顯著性的增加，不過在肺部的MLN中則無顯著性差異。除此之外，我們還發現肺部中IL-13和eotaxin-1 mRNA的量有顯著性的減少，血清中和肺部沖洗液的IgE也有降低的情況，顯示石斛多醣能藉由抑制免疫細胞在肺部中的聚集，減緩氣喘的現象。
根據之前實驗室的研究，發現山藥多醣有作為黏膜免疫佐劑的潛力，為了進一步確認這個現象，我們將山藥多醣和市售的感冒疫苗做混和後，以鼻點的方式做sensitization，犧牲後收取血清、肺部沖洗液和鼻腔沖洗液，以ELISA的方式檢測HA-specific IgG和 IgA的量是否有提升的作用。結果發現在給予山藥多醣
的組別對於提升IgG的效果最為明顯，在IgA部分也同樣具有提升效果，此結果表示山藥多醣具有做為黏膜免疫佐劑的能力，可以幫助產生血清和黏膜部位的IgG和IgA抗體，達到第一線的保護的作用。

The gut has a complex system to maintain the homeostasis in the battle of limiting inflammatory responses to commensal bacteria while retaining the ability to initiate protective adaptive immune responses to pathogens. Recently, some researches show that intestinal epithelial cells (IECs) may play key role in maintaining the homeostasis, for example, thymic stromal lymphopeitin (TSLP) produced by the IEC is capable of directing dendritic cells towards a Th2 response, and influencing the differentiation properties of monocytes into tissue macrophages. We’d like to develop a system to elucidate the bioactivity of polysaccharides from Chinese herbs (Ganoderma Lucidum, Astragalus mongholicus, Dendrobium huoshanense, Dioscorea opposita) using IEC-6 as a platform, and how they regulate intestinal immune system.
With the treatment of GaLuPS and AsMoPS，IEC-6 can up-regulate IL-6、TNF-α、TLR7、TLR9 gene expression, whileas DeCaPS can up-regulate TGF-刍 gene expression, which is known as immunosuppressive cytokine, leading to the proliferation of regulatory T cell.
In our in vivo OVA-induced asthma animal model, oral treatment with 30 and 90 mg/kg/day DeCaPS was shown to suppress asthma induced by active immunization and challenged with OVA. Here we show that DeCaPS can reduce pulmonary eosinophil recruitment and OVA-specific IgE in serum and bronchoalveolar lavage fluids (BALF), attenuate inflammation in lung, and decrease bronchial hyper-reactivity stimulated by methacholine. We also found that IL-13 and eosinophil-chemokine eotaxin（CCL11） mRNA from lung homogenates are lower in DeCaPS group. Furthermore, the population of CD4+CD25+ increases in intestinal lamina propira with the treatment of DeCaPS, whereas there is no difference in mediastinal lymph node in lung and spleen. According to these results, we demonstrate that DeCaPS has potential to attenuate asthma by suppressing the recruitment of eosinophil and Th2 cell in lung.
Our lab had previously demonstrated that DisPoPS has potential to be used as mucosal adjuvant, so we use BALB/c mice challenged with influenza vaccine mixed with DisPoPS intranasally to confirm this result. After sacrificing the mice at day 30, we found that DisPoPS was capable of inducing a significant increase in HA-specific total IgG and IgA from lung wash、nasal、vaginal wash and serum. Our results showed that DisPoPS is a good mucosal adjuvant.

目錄--------------------I
圖次--------------------II
縮寫表------------------IV
中文摘要----------------1
英文摘要----------------2
緒論--------------------3
試藥及材料--------------10
實驗方法----------------15
結果--------------------24
討論--------------------29
參考文獻----------------39
圖表 -------------------45

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