2013-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/713020摘要：Galectins 是一種 β-galactoside-binding 凝集素，它們具有不同的生物功能，如細胞黏著、細胞增生、細胞凋亡及調節發炎反應。其中 galectin-9不但是嗜伊紅白血球的趨化因子，細胞外的 galectin-9還可以經由 Tim-3/Gal-9的抑制性訊息傳導路徑來抑制 Th1細胞，對於免疫調節反應很重要。 IL-17在皮膚發炎反應中扮演著重要的角色，其最主要的來源為 T細胞及多形核白血球，尤以輔助型 T 細胞 Th17 為主要的分泌細胞。IL-17 和 IFN-γ可以加成作用於角質細胞，使其表面黏著分子ICMA-1及MHC分子表現增加，進而促進皮膚的發炎反應，如牛皮癬，接觸性皮膚炎等。在小鼠的接觸性過敏反應（一種過敏性接觸性皮膚炎的動物模式）中，IL-17基因剔除小鼠及給予 anti-IL-17單株抗體的小鼠其接觸性過敏反應較輕微。 在之前的計畫中我們藉由 Gal-9基因剔除的小鼠探討內生性 Gal-9的功能。我們的報告顯示 Gal-9基因剔除鼠的接觸性過敏反應比正常鼠來得輕，而將正常鼠及 Gal-9基因剔除鼠的 T細胞 adoptive transfer至正常鼠後，來自 Gal-9基因剔除鼠的 T細胞也產生較輕微的接觸性過敏反應。我們進一步用 anti-CD3/anti-CD28 抗體去刺激 T 細胞，發現 Gal-9 基因剔除鼠的 T細胞也比正常鼠的 T細胞分泌較少的 IFN-γ、IL-17及 IL-4；若是去刺激被活化的 T細胞（腹部皮膚塗抹 DNFB後 5天取出的 T細胞）則只有 IL-17有明顯的差異。由於先前有報告 IL-17缺乏的小鼠的接觸性過敏反應較輕微，因此我們推測 Gal-9 基因剔除鼠因產生較少的 IL-17導致較輕微的接觸性過敏反應。 本次計畫的目的是進一步研究內生性 galectin-9與 Th17分化及 IL-17製造的交互作用及相關性，我們的假說是在 Gal-9 基因剔除鼠中，其 Th17 分化及 IL-17 製造較少，且內生性galectin-9與細胞內的某蛋白質有交互作用以促進的 Th17分化。計畫工作時程如下： 第一年度我們將以不同的細胞激素去培養 T 細胞，測量其分化的 Th1/Th2/Th17 細胞的量,細胞激素的分泌量及其轉譯因子，以比較是否正常鼠比 Gal-9基因剔除鼠的 Th17細胞分化量較高。更進一步我們將使用Mass spectrometry及 two hybrid screening去找尋與 galectin-9交互作用的分子，以釐清 galectin-9與 Th17細胞分化的相關性。 第二年度我們將檢測此交互作用分子的功能以確認 galectin-9與 Th17細胞分化的詳細機轉。最後，我們將以多發性結節硬化症的動物模式來檢測是否 Gal-9 基因剔除鼠的反應與接觸性反應的表現一樣較輕微，以確認我們的研究的再現性。 經由此研究我們希望可以了解內生性 Gal-9在 Th17細胞分化的功能及影響，以進一步提供其治療相關疾病的可能機轉。<br> Abstract: Galectins (Gals) are members of a β-galactoside-binding animal lectin family which plays modulatory roles in diverse biological processes such as cell adhesion and proliferation, T cell apoptosis, and immunomodulation of inflammation. Gal-9 is well known for its chemotactic activity towards eosinophils and extracellular Gal-9 is also a negative regulator of Th1 cells through Tim-3/Gal-9 inhibitory signaling pathway, which is important in immune regulation. IL-17 is important in skin inflammation. The major cellular sources of IL-17 are T cells and granulocytes, while Th17 is the most important one. IL-17 acts synergistically with IFN-γ to induce adhesion molecules such as ICAM-1 or MHC antigens on keratinocytes, which results in an increased T cell mediated cytotoxicity for inflammatory skin diseases, such as psoriasis, contact dermatitis, etc. In contact hypersensitivity (CHS) response in mice, which is an animal model of allergic contact dermatitis, the IL-17 -/- mice or mice administered with anti-IL-17 mAbs showed attenuated CHS response. In our previous project, we showed that the CHS response in Gal-9 -/- mice and the CHS response by the adoptive transfer of T cells from Gal-9 -/- mice are less severe than that of Gal-9 +/+ mice. The T cells secreted less IFN-γ, IL-17 and IL-4 in Gal-9 -/- mice than in Gal-9 +/+ mice a under anti-CD3/anti-CD28 mAbs stimulation. But the T cells only secreted less IL-17 in Gal-9 -/- mice than in Gal-9 +/+ mice which were previously sensitized by DNFB for 5 days. Since it was reported that IL-17 -/- mice had less severe CHS response than IL-17 +/+ mice, we speculated that the less severe CHS response in Gal-9 -/- mice might be due to the less production of IL-17. Our aim of the present project is elucidate the correlation and interaction of endogenous galectin-9 and Th17 differentiation and IL-17 production. We hypothesize that the Th17 differentiation and IL-17 production is less in Gal-9 -/- mice and the endogenous galectin-9 may interact with proteins to promote the differentiation and signaling pathway. The working plan is as followings: In the first year, we will use the cytokine drive to check the differentiated Th1/Th2/Th17 cell amount and cytokine production (by ELISA and intracellular staining) and the transcription factor amount (T-bet/GATA-3/RORγt) to see if Th17 is less differentiated in Gal-9 -/- mice than in Gal-9 +/+ mice. Further, we will check the protein interactor of Gal-9 by mass spectrometry and two hybrid screening to find out the interaction. In the second year, we will check the function of the protein interactor to clarify the detail mechanism of Gal-9 interaction. We will also use the experimental autoimmune encephalomyelitis animal model to check if Gal-9 -/- mice are less severe than Gal-9 +/+ mice to validate our data in CHS model. Through this study, we hope that we can find out the endogenous function of galectin-9 in Th17 differentiation and the mechanism elucidated may give us new insight for treating allergic contact dermatitis and other Th17 mediated diseases.