臺灣博碩士論文加值系統

目前已知靈芝多醣體萃取物主要具有醣蛋白的部分並含有少量岩藻醣，而靈芝多醣體萃取物主要是透過類鐸受體調節各種免疫活性。我們首先發現靈芝多醣體萃取物可以使人類週邊血單核細胞表現更多CD25和FOXP3，更進一步發現，經靈芝多醣體萃取物處理的人類週邊血單核細胞具有免疫抑制的功效。接著我們觀察，在缺少抗原呈現細胞的情況下,靈芝萃取物同時加入抗CD3抗體和抗CD28抗體，可以刺激人類CD4+ T細胞增加CD25及FOXP3表現，且靈芝多醣體萃取物的刺激受到量和時間點的調控。本研究在靈芝多醣體萃取物和BLP(類鐸受體2)配體和LPS(類鐸受體4配體)的比較下發現，靈芝多醣體萃取物對於CD4+ T 細胞轉化為調節型T細胞有更強的反應。靈芝多醣體萃取物會刺激T輔助細胞(第一型T輔助細胞/第二型T輔助細胞)相關的細胞激素，例如：人類原CD4+ T細胞產生的介白素-2、干擾素-γ、介白素-4及介白素-10，但在轉化生長因子-β的產生沒有顯著變化。從我們的數據顯示靈芝多醣體萃取物同時在T細胞受體和CD28的訊息存在下，轉變為調節型T細胞的模式會於早期誘導產生介白素-2而晚期則產生介白素-10。另外，我們證明靈芝多醣體萃取物會刺激細胞毒性T淋巴細胞相關抗原4及CD69表現。我們使用特定的介白素-2中和抗體，顯示靈芝多醣體萃取物刺激T細胞受體活化CD4+ T細胞產生之介白素-2對於誘導調節型T細胞扮演重要角色。有趣的是，我們發現在短時間下使用特定的類鐸受體2和類鐸受體4中和抗體，可以降低靈芝多醣體萃取物刺激產生的CD25和FOXP3。這使我們假設類鐸受體2和類鐸受體4或其一對於調節型T細胞的表現扮演重要角色。最後，靈芝多醣體萃取物誘導產生的調節型T細胞除了具有免疫抑制的功效外，亦會使這群誘導產生的調節型T細胞增生。

An extract of Ganoderma lucidum (Reishi) polysaccharides (EORP), a well-characterized glycoprotein fraction containing fucose residues, exerts various immuno-modulation activities via Toll-like receptors (TLRs). First, we found EORP induce human PBMC express more CD25 and FOXP3. Moreover, EORP treated human PBMC have suppressive activity. Next we have investigate in the absence of antigen-presenting cells, EORP, together with anti-CD3 antibodies (αCD3) and anti-CD28 antibodies (αCD28), stimulates CD25 and FOXP3 expression in human primary CD4+ T cells in a time- and dose-dependent fashion. In this study, human CD4+ T cells could convert to CD4+CD25+ regulatory T cells (Tregs) in much response to EORP than BLP (TLR2 ligand) and LPS (TLR4 ligand). EORP stimulated Th (Th1/Th2)-related cytokines, such as IL-2, INF-��, IL-4 and IL-10 in human primary CD4+ T cells, but did not remarkable difference in TGF-β. Our data support a model of Tregs conversion in which EORP stimulation with TCR and CD28 signals induce IL-2 in early phase and IL-10 in late phase. In addition, we demonstrated that EORP stimulated cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) and CD69 expression. Using a specific neutralized IL-2 antibody, we showed that EORP induced IL-2 production in TCR-challenged CD4+ T cells play an important role in the induction of Tregs. We found that EORP stimulation of CD25 and FOXP3 expression could be completely reduced by short-term treatment with specific neutralized TLR2 and TLR4 antibodies, which suggests that TLR2 or TLR4, or both play important roles in the expression of Tregs. Finally, EORP induce Tregs have suppressive activity and also expansion of these CD4+CD25+ T cells.

中文摘要…………………………………………………………………1
Abstract…………………………………………………………………2
1. Introduction…………………………………………………………3
2. Materials and Methods…………………………………………6
2.1 Cell Preparation……………………………………………………………6
2.2 Reagents and antibodies……………………………………………………6
2.3 CD4+ T Cells Culture…………………………………………………………6
2.4 Induction and Determination of EORP-induced Cytokines in CD4+ T cells....7
2.5 Treg cell suppression assays…………………………………………………7
2.6 Flow Cytometry Analysis…………………………………………………8
3. Results………………………………………………………………9
3.1 EORP stimulates CD25 and FOXP3 expression in PBMC and modulation of the functions………………………………………………………………9
3.2 EORP stimulates primary CD4+ T cells more expression of CD25 and FOXP3, compared with LPS (TLR4 ligand) and BLP (TLR2 ligand)……………………9
3.3 EORP stimulated cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) and CD69 expression on CD25 and FOXP3 expression in human primary CD4+ T cells…………………………………………………………………10
3.4 EORP induced Tregs were suppressive…………………………………………11
3.5 EORP stimulates TGF-β, IL-2, INF-γ, IL-4 and IL-10 in human primary CD4+ T cells…11
3.6 Role of IL-2 in the EORP-stimulated CD25 and FOXP3 expression in human primary CD4+ T cells……………13
3.7 Role of Toll-like receptors (TLR2 and TLR4) upon EORP stimulated in human primary CD4+ T cells……………………………………………………………14
3.8 The effect of EORP induced CD4+CD25+ T cells proliferation in CD4+ T cell....15
4. Discussion……………………………………………………16
5. Reference………………………………………………………20
Figure legends……………………………………………25

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