江伯倫臺灣大學：免疫學研究所劉羽涵Liu, Yu-HanYu-HanLiu2007-11-292018-07-092007-11-292018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/63295介白質-10 (IL-10)是一種具有免疫抑制性的細胞激素，能夠抑制T細胞的活化及其功能，而對於調節性T細胞的分化也具有重要功能。介白質-10在許多自體免疫疾病之致病機轉中扮演的角色也益趨重要，例如全身性紅斑性狼瘡。若在週邊引起發炎反應的組織中給予介白質-10，可以降低發炎反應，並增強調節性T細胞之功能，則能預期對疾病症狀之舒緩，而作為細胞激素療法發展的學理根據。本論文著重於探討介白質-10的免疫調節功能對於抑制T細胞活化功能之機轉，我們觀察到給予介白質-10之T細胞株，會減少其IL-2之分泌量。我們也進一步研究介白質-10下游訊息的傳遞，並發現PPARγ的表現量在介白質-10刺激之下有明顯增加，同時，加入PPARγ的抑制劑會使介白質-10抑制T細胞IL-2分泌的情形獲得恢復。這些研究結果顯示，介白質-10對於T細胞的功能抑制可能是透過下游PPARγ的訊息傳遞，因此與PPARγ有關的訊息傳導路徑可能在自體免疫疾病中扮演著重要角色。為了進一步研究局部大量表現介白質-10與紅斑性狼瘡疾病舒緩之關聯，我們使用靈芝多醣體PSG，此成分能使樹突細胞與T細胞產生大量介白質-10，利用腹腔注射PSG的方式來治療狼瘡小鼠，治療結果發現小鼠周邊血中調節性T細胞之比例較高，腎絲球腎炎的情形較輕微，並有較長的壽命，說明了介白質-10在紅斑性狼瘡可能的療效。The immunosuppressive cytokine, interleukin-10, can inhibit the activation and effector functions of T cells. It also plays a key role in differentiation and function of a regulatory T cell subset. The role that IL-10 plays in the pathogenesis of many autoimmune diseases is striking, including systemic lupus erythematosus. Delivery of IL-10 cytokine alone in the inflammatory region to alleviate the severity of this disease, or enhance the function of regulatory T cells induced by the cytokine is the principle of tissue-specific cytokine treatment. In this study, we focused on the immune-modulatory effect of IL-10 in the activation of T cells. Decreased production of IL-2 was observed in different T cell lines when cultured with IL-10. To further study the inhibitory mechanisms of IL-10 on T cells, we examined the signaling pathways downstream of IL-10 and found the increased PPARγ expression was induced by IL-10 treatment. In addition, the inhibition of IL-2 secretion by IL-10 could be partially restored by PPARγ antagonist, GW9662. Our results suggest the inhibitory effect on T cells by interleukin-10 is partly through their downstream PPARγ. This molecule and its associated signal pathways should be taken into consideration for its potential of being new target in the development of immunotherapy against SLE. To further understand the effects of IL-10 on the disease development of SLE in vivo, we intraperitoneally injected PSG to NZB/W F1 mice to induce IL-10 production. After treatment, the mice have higher percentage of Treg population, lower glomerulonephritis, and greater survival tendencies. This suggests the potential role of IL-10 in the treatment of murine lupus.謝誌…i 英文摘要…iii 中文摘要…v Chapter I. General Introducion 1.1 Background…2 1.1.1 Systemic lupus erythematosus…2 1.1.2 Murine model of lupus: NZB/W F1 mice…7 1.1.3 Interleukin-10…8 1.1.4 The role of interleukin-10 in SLE…9 1.1.5 The relationship of interleukin-10 and regulatory T cell function…10 1.1.6 Lentiviral vector…11 1.1.7 Peroxisome proliferator-activated receptor-gamma (PPARγ) …12 1.1.8 Polysaccharide components of Ganoderma lucidum (PSG) …14 1.2 Specific aims…15 Chapter II. Materials and Methods 2.1 Reagents…17 2.2 Cell lines…20 2.3 Plasmid construction…21 2.4 Transfection of COS-1 cells…22 2.5 Preparation of lentiviruses…22 2.6 Detection of cytokine expression by ELISA…23 2.7 Separation of splenic T cells by nylon wool…24 2.8 Stimulation of T cells…24 2.9 Letivirus transduction of splenic T cells…25 2.10 Detection of protein levels 2.10.1 SDS-PAGE…25 2.10.2 Western blot…26 2.11 Mice…27 2.12 In vivo treatment of PSG in NZB/W F1 mice…27 2.13 Examination of immunopathology 2.13.1 Detection of autoantibodies…28 2.13.2 Determination of renal pathology…29 2.14 Statistical analysis…30 Chapter III. Results 3.1 The inhibitory effect of IL-10 on T cell lines…32 3.2 Construction of pTYlinker-mIL10 plasmid for lentiviral delivery of mouse IL-10 gene 3.2.1 Construction of pTYlinker-mIL10 plasmid…32 3.2.2 IL-10-secreting capability of pTYlinker-mIL10 transfected cells…33 3.2.3 IL-10 production by lenti-mIL10 virus tranduced cells…33 3.2.4 Lenti-mIL10 virus-tranduction in mouse splenic T cells…34 3.3 The role of PPARγ in the inhibitory effect of IL-10 3.3.1 Ligand induced PPARγ expression in Jurkat cells…35 3.3.2 IL-10 induced PPARγ expression in human and mouse T cell lines…35 3.3.3 PPARγ antagonist reversed the inhibitory effect of IL-10 in human and mouse T cell lines…36 3.4 Application of PSG as an alternative way of local IL-10 induction for lupus treatment 3.4.1 PSG induced IL-10 production by dendritic cells of NZB/W F1 mice in vitro…36 3.4.2 PSG induced maturation of BMDC by enhancing the expression of DC surface markers and costimulatory molecules in vitro…37 3.5 PSG-treated lupus mice 3.5.1 Peripheral CD4+CD25+FoxP3+ regulatory T cells were increased after PSG treatment…38 3.5.2 Levels of autoantibodies in sera were no difference under PSG treatment…38 3.5.3 Clinical course and pathological changes of kidney…39 3.5.4 Prolonged survival of PSG-treated mice…40 Chapter IV. Discussion and perspectives…41 Figures…47 References…701414888 bytesapplication/pdfen-US介白質-10全身性紅斑性狼瘡T細胞自體免疫過氧化小體增殖物活化受體靈芝多醣體Interleukin-10Systemic lupus erythematosusT cellAutoimmunePeroxisome proliferator-activated receptor(PPAR-gamma)The polysaccharide components of Gernderma lucidum(PSG)otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63295/1/ntu-96-R93449005-1.pdf