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研究生:蕭惠文
研究生(外文):Huey-Wen Hsiao
論文名稱:Deltex1在淋巴細胞發育與T細胞活化中之角色探討
論文名稱(外文):The Role of Deltex1 in Lymphocyte Development and T cell Activation
指導教授:賴明宗賴明宗引用關係賴明宗賴明宗引用關係
指導教授(外文):Ming-Zong LaiMing-Zong Lai
學位類別:博士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:131
中文關鍵詞:T細胞活化自體免疫
外文關鍵詞:DeltexNotchT cell activation
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Deltex1是Notch受體的結合蛋白,且其RF (RING finger)區塊具有E3泛素連接脢活性。在哺乳動物的研究中,DTX1的功能尚不明確。在本論文中發現DTX1可透過蛋白脢體的分解途徑抑制活化型Notch1受體的產生。為探討缺失DTX1對淋巴細胞發育的影響以及DTX1在活體內可能具有的生理意義,我們建立了基因標的Dtx1 exon 3的Dtx1剔除 (Dtx1-/-)小鼠。結果觀察到Dtx1-/-小鼠中,胸腺與脾臟中T細胞發育為正常,骨髓中B細胞發育也無異常,然而脾臟中MZ B細胞與腹腔中的B1細胞群減少一半。
先前研究中,發現大量表現DTX1可抑制T細胞活化,且經由RF區塊造成MEKK1(C)的蛋白分解。本研究亦可觀察到Dtx1-/-T細胞活化後的細胞增生、激素分泌與MAPK活性皆增強許多。此外,我們發現缺失WWE區塊或RF區塊的DTX1突變型仍可抑制T細胞活化後的IL-2產生。進一步我們發現DTX1可調控Gadd45�猁漯穛{,Gadd45�狺w知可抑制JNK活化。且DTX1不需WWE區塊與E3連接脢活性即可活化Gadd45b啟動子。顯示DTX1抑制T細胞活化並非單一機制,除了利用本身E3連接脢活性使MEKK1(C)分解,也可透過不依賴WWE區塊與RF區塊的機制活化Gadd45b的表現。進一步我們分析高齡的Dtx1-/-小鼠,結果觀察到血液中有較多的自體抗體的存在,在腎絲球中有自體免疫複合物沉積,且在肺臟和肝臟出現淋巴細胞的大量聚集。綜合以上研究結果,我們發現DTX1可參與在MZ B與B1細胞的發育分化,且DTX1對於調控T細胞耐受性與自體免疫反應中具有重要的功能。
Deltex1 (DTX1) is identified as a Notch-interacted molecule and a RING finger (RF) ubiquitin ligase. The function of DTX1 in mammals remains unclear. In this study, we found that overexpression of DTX1 led to degradation of active form Notch1 through proteasome pathway. To further investigate the physiological function of DTX1 in Notch-regulated lymphocyte development and activation, Dtx1 exon3 was targeted to generate Dtx1-/- mice. Dtx1-/- mice displayed normal T cell development in thymus and spleen and normal B cell development in bone marrow. Splenic marginal zone (MZ) B cell and peritoneal B1 cell population were reduced in Dtx1-/- mice. We have previously found that overexpression of DTX1 suppresses T cell activation and promotes MEKK1 degradation. Consistent with the negative role of DTX1 in T cell activation, we found that T cell proliferation, cytokine production and MAPK activation were enhanced in Dtx1-/- T cells. In addition, DTX1 with deletion of WWE domain or RF domain still suppressed T cell activation. We identified a new suppressive mechanism of DTX1. DTX1 upregulated the expression of Gadd45b, which was an inhibitor of JNK activation. Furthermore DTX1 activated Gadd45b promoter in the absence of RF domain or WWE domain. Therefore, DTX1 may inhibit T cell activation independent of E3 ligase activity or Notch-binding ability. The in vivo function of DTX1 was further demonstrated by the enhanced autoantibody production, elevated immune complex deposition in kidney, and increased leukocyte infiltration in lung and liver in aged Dtx1-/- mice. Together these data suggest that DTX1 plays a role in the development of MZ B and B1 B cells, and DTX1 is critical in the maintenance of T cell tolerance and control of autoimmunity.
摘要……………………………………………………………………i
ABSTRACT……………………………………………………………ii
目錄…………………………………………………………………iv
第一章 緒論
1. 淋巴細胞的發育與分化…………………………………1
1.1. 胸腺T細胞的發育
1.2. 周邊T細胞的發育與分化
1.3. 骨髓B細胞的發育
1.4. 周邊B2細胞的發育與分化
1.5. B1細胞
1.6. B細胞分化與B細胞受體活化
2. Notch受體………………………………………………6
2.1. Notch受體的結構
2.2. Notch受體的訊息傳遞
2.3. Notch受體訊息傳遞對淋巴細胞發育的影響
3. DTX1……………………………………………………10
3.1. DTX1基因結構及其轉譯出之功能性區段
3.2. DTX與Notch受體訊息傳遞
3.3. DTX1對淋巴細胞發育的影響
3.4. DTX1對T細胞活化的影響
4. Gadd45b……………………………………………15
4.1. Gadd45b的轉錄調控
4.2. Gadd45b的訊息傳遞
4.3. Gadd45b在T細胞的功能
5. 研究方向與目的 …………………………………17
第二章 材料與方法
1. 質體構築……………………………………………19
2. 建立選擇性Dtx1基因剔除小鼠………………………21
3. 細菌培養……………………………………………28
4. 質體篩檢與gDNA製備…………………………28
5. RNA的純化與分析……………………………30
6. 南方墨點法……………………………………………31
7. 細胞純化與培養…………………………………………33
8. 細胞轉染與報告載體活性分析…………………………35
9. 反轉錄病毒的製備與感染………………………………36
10. 淋巴細胞的活化與增生…………………………………37
11. T細胞的活化與細胞激素的產量分析…………………38
12. 流式細胞儀分析………………………………………40
13. 免疫墨點法……………………………………………41
14. 自體抗體測量…………………………………………43
15. 小鼠病理切片觀察……………………………………44
第三章 實驗結果
1. DTX1抑制Notch受體的訊息傳遞……………………47
1.1. DTX1抑制N1-IC對Hes5啟動子的轉錄活性
1.2. DTX1抑制N1-IC的表現
2. 剔除Dtx1對淋巴細胞發育的影響………………………48
2.1. 建立選擇性條件式Dtx1基因剔除小鼠
2.2. Dtx1基因剔除小鼠的遺傳鑑定及表現分析
2.3. 剔除Dtx1造成胸腺和脾臟的細胞數量增加
2.4. 剔除Dtx1不影響T細胞凋亡
2.5. 剔除Dtx1不影響胸腺中T細胞的發育
2.6. 剔除Dtx1不影響脾臟和淋巴結中T細胞的發育
2.7. 剔除Dtx1不影響骨髓中的細胞數量和B細胞發育
2.8. 剔除Dtx1造成MZB細胞族群減少
2.9. 剔除Dtx1造成腹腔內B1細胞族群減少
3. 剔除Dtx1增強B細胞受體的活化……………………56
3.1. 剔除Dtx1增加B細胞活化中蛋白質的磷酸化
3.2. 剔除Dtx1增加B細胞活化後的增生
3.3. 剔除Dtx1不影響B細胞活化造成的細胞凋亡
4. 剔除Dtx1增強T細胞的活化…………………………57
4.1. 剔除Dtx1增加T細胞活化後的增生與分裂
4.2. 剔除Dtx1不影響T細胞活化造成的細胞凋亡
4.3. 剔除Dtx1增加T細胞活化中IL-2、IFN-r和IL-4的產生
4.4. 剔除Dtx1增加T細胞活化中MAPK的活化
5. DTX1引發Gadd45b的表現……………………………60
5.1. DTX1dRF可抑制T細胞活化中IL-2的產生
5.2. DTX1dRF抑制T細胞活化中JNK的磷酸化但不影響p38 MAPK的磷酸化
5.3. DTX1dN可抑制T細胞活化中IL-2的產生
5.4. Gadd45b抑制T細胞活化中IL-2的產生
5.5. DTX1引發Gadd45b的RNA及蛋白質表現
5.6. DTX1活化Gadd45b啟動子的轉錄活性
6. Dtx1剔除小鼠易產生自體免疫………………………63
6.1. 年老的Dtx1剔除小鼠的肺臟和肝臟有大量的淋巴細胞滲透
6.2. 年老的Dtx1剔除小鼠的腎臟出現免疫球蛋白的沉積
6.3. Dtx1剔除小鼠的血液中有較多的自體抗體
6.4. 剔除Dtx1增加Th17細胞的分化
第四章 結果討論……………………………………………67
圖表………………………………………………………74
參考文獻………………………………………………120
發表著作…………………………………………………125
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