指導教授：江伯倫臺灣大學：免疫學研究所楊智傑Yang, Chih-ChiehChih-ChiehYang2014-11-262018-07-092014-11-262018-07-092014http://ntur.lib.ntu.edu.tw//handle/246246/262026　　Treg細胞有抑制免疫反應的功能，在免疫系統中扮演重要角色。過去研究指出，當CD4+ T細胞受到細胞激素刺激時，可以轉變為CD4+ CD25+ T細胞，並且有免疫抑制功能，稱作為iTreg細胞。最近我們的研究發現，以B2細胞作為抗原呈獻細胞可以誘導CD4+ T細胞成為類似Treg的細胞，我們稱之為Treg-of-B2細胞，而對於Treg-of-B2細胞的免疫抑制機轉目前並不清楚。動物凝集素家族中的半乳糖凝集素是最近免疫研究上一個重要的議題，目前發現了15種的半乳糖凝集素，但大部分的功能並不清楚。半乳糖凝集素在免疫細胞中參與了許多反應，例如半乳糖凝集素-2可以誘發CD8+ T細胞的細胞凋亡，但是半乳糖凝集素-3反而是抑制細胞凋亡的發生。本研究探討半乳糖凝集素在Treg-of-B2細胞的免疫抑制中的影響，首先收集活化的Treg-of-B2細胞，分析半乳糖凝集素的RNA表現，結果發現半乳糖凝集素-2有高量的表現，並且同時也可以在細胞培養液中測到半乳糖凝集素-2的表現。本次研究也同時分析Tr1細胞的半乳糖凝集素RNA表現，但並沒有任何表現。接著發現Treg-of-B2細胞執行免疫抑制功能時，不一定需要細胞接觸，在加入乳糖阻礙半乳糖凝集素作用，發現Treg-of-B2細胞的免疫抑制功能有顯著的下降。CD4+ T細胞的增生實驗中加入半乳糖凝集素-2發現半乳糖凝集素-2可抑制CD4+ T細胞的增生，因此透過本次研究可知半乳糖凝集素-2參與在Treg-of-B2細胞的免疫抑制功能中。Regulatory T cells (Treg cells) have an ability to suppress immune response. Under particular condition, naive CD4+ T cell could differentiate into inducible Treg (iTreg) cells with regulatory functions. In the past few years, some in vitro experiments have shown that splenic B220+ B2 cells could convert naive CD4+ T cells into induced Treg cells (referred to as Treg-of-B2 cells). Treg-of-B2 cells had immune suppressive ability like nTreg cells, but the mechanism remained unclear. To date, 15 galectins have been identified in mammals, only galectin-1, -2, -3, -4, -7, -8, -9, -12 identified in mice. Recent studies have shown that several galectins (including galectin-1, -2, -3, -7, -8, -9, and -12) induced apoptosis in T cell. Galectin-1 was a key effector molecule in the regulation function of CD4+CD25+ T cells. Galectin-2 induced activated CD8+ T cells apoptosis. Galectin-3 exerted several functions such as inhibition of apoptosis, promotion of cell growth, and regulation of TCR signaling. The aim of our study was to investigate the relationship between galectins and the regulatory function of Treg-of-B cells. First, expression profile of galectins in activated Treg-of-B2 cells and activated Tr1 cells had been screened. We found that activated Treg-of-B2 cells had higher galectin-2 expression but Tr1 cells did not. Second, the suppressive function of Treg-of-B2 cells did not need cell-cell contact. By adding lactose and transwell system in suppressive assay, we found that galectin-2 might be a factor in the function of Treg-of-B2 cells. We confirmed the role of galectin-2 in the proliferation of CD4+ CD25- T cells by adding recombinant galectin-2 in culture medium and we found that the proliferation of CD4+ CD25- T cells was decreased. Our finding clarify that a part of suppressive ability of Treg-of-B2 cells was galectin-2 secretion.第一章 緒論 ................................................................................... 1 第一節 背景 .................................................................................. 2 1. B2細胞（B2 cell）......................................................................... 2 2. T細胞（T cell）........................................................................... 3 3. 調節性T細胞（Regulatory T cell, Treg）..................................................... 4 4. 誘發性調節性T細胞（Induced regulatory T cell, iTreg）....................................... 5 5. 第ㄧ型調節性T細胞（Type 1 regulatory T cell, Tr1）.......................................... 5 6. Treg-of-B2細胞 .......................................................................... 6 7. 半乳糖凝集素（Galectins）.................................................................. 6 8. 半乳糖凝集素與調節性T細胞 ................................................................... 8 第二節 研究動機與目的 .......................................................................... 9 第二章 實驗方法及材料 ........................................................................... 10 第一節 初代細胞之收集 ...........................................................................11 第二節 Treg-of-B2細胞培養.......................................................................11 第三節 第一型調節性T細胞培養..................................................................... 12 第四節 細胞分析 ............................................................................... 12 第五節 免疫抑制試驗 ............................................................................ 12 第六節 乳糖中和試驗 ............................................................................ 13 第七節 RNA萃取................................................................................ 13 第八節 逆轉錄聚合酶鏈鎖反應 ...................................................................... 14 第九節 定量即時聚合酶鏈鎖反應 ..................................................................... 14 第十節 西方墨點法 .............................................................................. 14 1. 蛋白質萃取................................................................................. 14 2. 蛋白質濃度測定.............................................................................. 15 3. 膠體電泳................................................................................... 15 4. 轉印...................................................................................... 15 5. 底片呈色................................................................................... 15 第十一節 Transwell系統.......................................................................... 16 第十二節 統計分析 ............................................................................... 16 第十三節 實驗相關材料 ............................................................................ 16 1. 細胞培養液及緩衝液........................................................................... 16 2. BD IMagTM system.......................................................................... 17 3. Mitogens、單株抗體及細胞激素.................................................................. 17 4. 細胞分析.................................................................................... 18 5. 免疫抑制試驗................................................................................. 19 6. RNA 萃取.................................................................................... 19 7. 逆轉錄聚合酶鏈鎖反應........................................................................... 19 8. 定量即時聚合酶鏈鎖反應......................................................................... 20 9. Cell lysis ................................................................................ 21 10. 西方墨點法.................................................................................. 21 11. Transwell系統.............................................................................. 22 第三章 結果 ...................................................................................... 23 第一節 使用流式細胞儀分析BD IMagTM system分選出的脾臟免疫細胞........................................... 24 第二節 使用流式細胞儀分析Treg-of-B2細胞與第一型調節性T細胞的細胞標記....................................... 24 第三節 測試Treg-of-B2細胞與第一型調節性T細胞的免疫抑制功能............................................... 25 第四節 測量活化的Treg-of-B2細胞、第一型調節性T細胞與自然調節性T細胞的半乳糖凝集素基因表現...................... 25 第五節 以西方墨點法偵測細胞培養液中的半乳糖凝集素-2...................................................... 26 第六節 將乳糖加入Treg-of-B2細胞抑制CD4+ CD25- T細胞的實驗............................................. 27 第七節以transwell系統瞭解Treg-of-B2細胞對於CD4+ CD25- T細胞的免疫抑制機轉............................... 27 第八節 將半乳糖凝集素-2加入CD4+ CD25- T細胞的增生實驗.................................................. 28 第九節 將乳糖加入Treg-of-B2細胞抑制CD8+ T細胞的實驗................................................... 28 第十節 以transwell系統瞭解Treg-of-B2細胞對於CD8+ T細胞的免疫抑制機轉.................................... 29 第十一節 將半乳糖凝集素-2加入CD8+ T細胞的增生實驗....................................................... 30 第四章 討論 ....................................................................................... 31 第五章 參考文獻 .................................................................................... 36 圖目錄 圖 一、使用流式細胞儀分析由BD IMagTM system分選出的脾臟免疫細胞........................................... 48 圖 二、使用流式細胞儀分析Treg-of-B2細胞與第一型調節性T細胞的細胞標記........................................ 49 圖 三、測試Treg-of-B2細胞與第一型調節性T細胞的免疫抑制功能................................................ 51 圖 四、測量活化細胞的半乳糖凝集素基因表現 ............................................................... 52 圖 五、以西方墨點法偵測細胞培養液中的半乳糖凝集素-2....................................................... 53 圖 六、將乳糖加入Treg-of-B2細胞抑制CD4+ CD25- T細胞的實驗............................................... 54 圖 七、以transwell系統瞭解Treg-of-B2細胞對於CD4+ CD25- T細胞的免疫抑制機轉............................... 56 圖 八、將半乳糖凝集素-2加入CD4+ CD25- T細胞的增生實驗................................................... 57 圖 九、將乳糖加入Treg-of-B2細胞抑制CD8+ T細胞的實驗.................................................... 58 圖 十、以transwell系統瞭解Treg-of-B2細胞對於CD8+ T細胞的免疫抑制機轉 .................................... 60 圖 十一、將半乳糖凝集素-2加入CD8+ T細胞的增生實驗........................................................ 611308408 bytesapplication/pdf論文使用權限：同意有償授權(權利金給回饋本人)半乳糖凝集素-2調節性T細胞Treg-of-B2細胞thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262026/1/ntu-103-R01449013-1.pdf