江伯倫臺灣大學：免疫學研究所徐慰婷Hsu, Wei-TingWei-TingHsu2007-11-292018-07-092007-11-292018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/63330最近研究顯示調節性T細胞的不正常可能在自體免疫疾病致病過程中扮演重要角色。一群由胸腺產生持續表現CD25分子的CD4+ 細胞具有免疫抑制能力，去除掉這群CD4+CD25+ T細胞將導致器官特異性自體免疫疾病發生。紅斑性狼瘡是一種慢性全身性的自體免疫疾病，特徵為會產生多種對抗核內抗原的自體抗體。到目前為止，調節性T細胞是否參予這個疾病的致病過程並不清楚。為了去探討這個問題，我們測定狼瘡小鼠(NZB/NZW F1) 與正常小鼠(DBA2/NZW F1) CD4+CD25+ T細胞的比例與相關重要基因表現。結果顯示CD4+CD25+ T細胞的比例在年輕狼瘡小鼠體內較正常小鼠低；但到發病晚期，狼瘡小鼠的CD4+CD25+ T細胞逐漸增加，甚至比正常小鼠高。並且在年輕的狼瘡小鼠中，我們可以偵測到核小體特異性T細胞的存在。我們推測在狼瘡小鼠的調節性T細胞與自體反應T細胞數目的不平衡，可能為導致疾病發展的因素之ㄧ。進一歩研究發現，兩種小鼠的CD4+CD25+ T細胞其Foxp3 與TGF-beta基因表現量差不多，但狼瘡小鼠的CD4+CD25+ T細胞與CD4+CD25- T細胞IL-10的表現都較正常小鼠來的高。由體外抑制細胞增生實驗得到，狼瘡小鼠的CD4+CD25+ T細胞仍具有免疫抑制能力。另一方面，我們利用抗CD25抗體去除掉正常小鼠的CD4+CD25+ T細胞，接著用凋亡小體處理過的樹突細胞破壞其免疫耐受性，並偵測體內自體抗體的產生。我們發現去除掉CD4+CD25+ T細胞的小鼠組別會產生較高的抗雙股/單股去氧核醣核酸抗體。這個結果顯示缺乏CD4+CD25+ T細胞的情況下，會使得調節自體抗體產生的機制受到破壞，證明CD4+CD25+ T細胞在自體免疫反應調控上可能扮演著重要的角色。Recent studies have suggested that the dysfunction of regulatory T cells might play a critical role in the pathogenesis of autoimmune diseases. A subset of thymus-derived CD4+ cells that constitutively express CD25 exhibits immune suppressive activity. Depletion of CD4+CD25+ T cells has been shown to cause organ-specific autoimmune diseases. Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disease characterized by the production of antibodies to components of the cell nucleus. At present, it is unknown if regulatory T (Treg) cells are involved in the pathogenesis of SLE. In order to address this question, we assayed the frequency of CD4+CD25+ T cells and measured the related gene expression levels in CD4+CD25+ T cells isolated from both lupus mice (NZB/NZW F1) and normal mice (DBA2/NZW F1). The data showed that the frequency of CD4+CD25+ T cells in young lupus mice was lower than that in normal mice. But in old mice, the frequency of CD4+CD25+ T cells increased and was even higher than that in age matched normal mice. Nucleosome-specific T cells can also be detected in young lupus mice. Therefore, we proposed that the imblalance of regulatory T cells in lupus mice might cause the development of this disease. Furthermore, the Foxp3 and TGF-beta expression of CD4+CD25+ T cells were very similar between both groups of mice. In contrast, the IL-10 expression of CD4+CD25+ T cells isolated from lupus mice was higher than that of normal mice. In in vitro suppression assays, CD4+CD25+ T cells of lupus mice were found to exert suppressive activities as well. Further, we depleted CD4+CD25+ T cells with anti-CD25 antibodies in non-autoimmune mice and broke the tolerance with dendritic cells pulsed with apoptotic cells for the follow up of autoantibody levels. We found that the mice depleted CD4+CD25+ T cells had higher titer of auto-anti-ds/ss DNA antibodies compared with that of control groups. The finding indicated that CD4+CD25+ T cell activities might be involved in the regulating mechanism of autoantibody production. Although more studies are needed, CD4+CD25+ T cells might play a critical role in the regulation of autoimmune response in murine lupus.論文口試委員審定書…………………………………………………….i 授權書……………………………………………………………………ii 誌謝……………………………………………………………………...iv 中文摘要…………………………………………………………………v 英文摘要………………………………………………………………..vii 縮寫對照表……………………………………………………………...ix 目錄……………………………………………………………………...xi 第一章 緒論 1.1 紅斑性狼瘡之背景介紹…………………………………………..1 1.2 核小體與核小體特異性T細胞在紅斑性狼瘡中扮演的角色 1.2.1 核小體與抗核小體抗體…………...…………………………..6 1.2.2 核小體特異性T細胞在紅斑性狼瘡中扮演的角色……….....8 1.3 CD4+CD25+調節性T細胞 1.3.1 CD4+CD25+調節性T細胞……………………………………..9 1.3.2 CD4+CD25+調節性T細胞與自體免疫疾病的關係…………11 1.4 紅斑性狼瘡的動物模式---NZB/NZW F1小鼠…………………12 第二章 研究動機與目的 2.1 研究動機…………………………………………………………15 2.2 研究目的…………………………………………………………17 第三章 實驗材料與方法 3.1 實驗用小鼠………………………………………………………18 3.2 凋亡小體之取得…………………………………………………18 3.3 骨髓衍生性樹突細胞培養………………………………………19 3.4 細胞純化…………………………………………………………21 3.5 T細胞分裂增生實驗……………………………………………..23 3.6 ELISPOT實驗及影像分析………………………………………24 3.7 採小鼠眼窩血分析細胞比例……………………………………25 3.8 螢光流體計數儀…………………………………………………26 3.9 純化mRNA及反轉錄成cDNA…………………………………27 3.10 以real-time PCR定量基因表現…….…………………………28 3.11 抑制細胞增生實驗……………………………………………..28 3.12 利用體內實驗(in vivo)破壞正常小鼠(DBA/NZW F1)的耐受性 …………………………………………………………………...29 3.13 ELISA檢測抗DNA抗體………………………………………30 3.14 偵測尿蛋白……………………………………………………..31 第四章 實驗結果 4.1 偵測核小體特異性T 細胞的數目比例 4.1.1 培養樹突細胞………………………………………………...32 4.1.2 比較NZB/NZW F1小鼠與DBA/NZW F1小鼠的核小體特異 性CD4+ T細胞增生反應之差異…………………………….32 4.1.3 偵測NZB/NZW F1小鼠的核小體特異CD4+ T細胞比例….33 4.2 偵測CD4+CD25+ T細胞的比例和與其抑制弁酮袺鰝滌穧]表 現 4.2.1 分析小鼠中CD4+CD25+ T細胞的比例……………………..34 4.2.2 分析小鼠中CD4+CD25+ T細胞Foxp3、IL-10及TGF-β基 因的表現……………………………………………………...35 4.3 分析發病的狼瘡小鼠其CD4+CD25+和CD4+CD25-兩群細胞表 現的Th1/Th2細胞激素趨勢……………………………………..36 4.4 體外實驗(in vitro)分析狼瘡小鼠CD4+CD25+ T細胞的抑制弁赨r …………………………………………………………………...37 4.5 體內實驗(in vivo)證明CD4+CD25+ T細胞在自體免疫反應中扮 演重要角色……………………………………………………….38 第五章 討論與結論…………………………………………………….41 圖表……………………………………………………………………...47 參考文獻………………………………………………………………...67 附錄……………………………………………………………………..77655079 bytesapplication/pdfen-US紅斑性狼瘡調節性T細胞狼瘡小鼠SLEregulatory T cellBWF1otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63330/1/ntu-93-R91449006-1.pdf