臺灣博碩士論文加值系統

摘要
Deltex最初是在果蠅中所發現，參與在影響細胞命運的Notch訊號途徑中。而哺乳類Deltex (DTX)家族相關的研究目前仍尚未完全明瞭。研究已知Deltex1在T細胞活化中是一個負調控角色，Deltex1 (DTX1)缺陷會導致小鼠出現自體免疫現象。而Deltex2 (DTX2)，在氨基酸序列上與DTX1具有高相似度，但是對於 DTX2的生理功能仍不清楚。實驗室先前研究發現，DTX2缺陷會減弱T細胞活化，但會增強巨噬細胞活化。在巨噬細胞中DTX1的表現量極低，而DTX2則是大量表現。因此在本論文研究中要探討DTX2在巨噬細胞活化中所扮演的確切角色為何。我們發現不同的Toll-like receptor (TLR)-ligand刺激下，相對於對照組巨噬細胞，DTX2缺陷小鼠巨噬細胞會產生較高的TNF-α、IL-6 及IL-1β。也表現較高的第一型干擾素信使核醣核酸。在人類單核球細胞THP-1下調DTX2，同樣觀察到在TLR刺激下有較高的TNF-α 和IL-6產生。DTX2基因剔除巨噬細胞在TLR刺激下有較顯著的NF-κΒ活化，包括IKK/IκΒα 磷酸化及IκΒα降解。在找尋DTX2於NF-κΒ訊息路徑中的目標蛋白時，我們發現DTX2會和TRAF6 (Tumor necrosis factor associated factor 6)結合，但不會和NEMO (NF-kappa-B essential modulator), TAK1 (Transforming growth factor beta-activated kinase 1)結合。在293T細胞中過度表現DTX2，不會影響TRAF6的表現量，但大量表現DTX2會減弱TRAF6主導的κB-Luciferase活化。進一步利用免疫沈澱法，在DTX2缺失巨噬細胞中經TLR2刺激後，會看到TRAF6與NEMO, IKKβ出現較強且久的結合。此外， DTX2會藉由Ring-Finger motif及proline-rich region 兩個區段與TRAF6結合。並且，我們發現引發小鼠敗血性休克後，DTX2缺失小鼠比對照組有較高死亡率，顯示DTX2在生理情況下的抗發炎功能。
此研究結果顯示，DTX2負參與調控巨噬細胞活化，藉由和 TRAF6結合，會延遲且抑制NEMO, IKKβ與TRAF6結合， 進而減弱TLR刺激下NF-κΒ活化抑制巨噬細胞活化。DTX2缺失導致嚴重發炎。

Abstract
Deltex is first identified in Drosophila as a mediator of Notch signaling. The role of Deltex family genes in mammalians is incompletely understood. Deltex1 is a negative regulator of T cell activation and its deficiency leads to autoimmunity. Deltex2 (DTX2) shares sequence similarity with Deltex1, but the physiological function of Deltx2 is still unknown. Preliminary data from our lab found Dtx2-deficiency led to attenuate T cell activation, yet increased macrophage activation. While Deltex1 is expressed at very low level, DTX2 is abundantly expressed in macrophages. In this study, we plan to identify the exact role of DTX2 in macrophage activation. Dtx2-knockout enhanced different Toll-like receptors (TLRs) ligands-induced secretion of TNF-α, IL-6 and IL-1β in macrophages. Increased the production of TNF-α and IL-6 were also observed in DELTEX2-knockdown THP-1 cells. The expression of type I interferon mRNA level was also higher in activated Dtx2-knockout macrophages. Dtx2-deficient macrophages exhibited enhanced Pam3CSK4-induced NF-κΒ activation, including IKK activation, ΙκΒα phosphorylation and IκBα degradation. In search for possible DTX2 target involved in NF-κΒ activation, we found that DTX2 interacted with tumor necrosis factor associated factor 6 (TRAF6) but not two effector proteins participating in NF-κΒ signaling, TAK1 and NEMO. Even through DTX2 overexpression did not affect TRAF6 protein level in 293T cells, DXT2 overexpression decreased TRAF6 mediated κΒ-Luciferase activation. In immunoprecipitation analysis the association of TRAF6 with NEMO and IKKβ are higher in Dtx2-deficient macrophages than WT macrophages after Pam3CSK4 stimulation . We further mapped the TRAF6-binding domain of DTX2 Ring-Finger motif and proline-rich region. Upon intraperitoneal administration of LPS, Dtx2-deficiency mice displayed higher mortality than wild-type controls. Together, our results suggest that DTX2 inhibits NF-κΒ activation by interacting with TRAF6 and decrease the NEMO/IKKβ/TRAF6-complex association. DTX2 is a negative regulator of macrophage activation and its deficiency leads to hyper-inflammation.

目錄

ABSTRACT I
摘要 III
縮寫表 V
目錄 VII
圖表目錄 IX
第一章 簡介 1
1. NF-κB 1
2. Deltex 4
3. 研究方向與目的 6
第二章 材料與方法 7
1. 細胞純化與培養 7
2. Lentivirus 反轉錄病毒製備與感染 8
3. DNA轉染 9
4. 西方點墨法 10
5. 冷光酵素活性測定（Luciferase assay） 12
6. ELISA分析 12
7. RNA萃取 13
8. 即時聚合酶鏈式反應（Quantitative PCR） 14
第三章 實驗結果 15
1. DTX2缺失會增強TLR刺激小鼠骨髓巨噬細胞產生之TNF-α、IL-及IL-1β 15
2. DTX2缺失會增加TLR刺激下小鼠腹腔巨噬細胞第一型干擾素表現 15
3. 建立下調DTX2之THP-1細胞株 16
4. 下調DTX2增強TLR活化THP-1 細胞產生之TNF-α、IL- 16
5. DTX2缺失增強小鼠骨髓巨噬細胞於TLR活化下IKK/κα磷酸化 17
6. DTX2缺失些微影響TLR活化小鼠骨髓巨噬細胞所引發ERK/Akt磷酸化 17
7. DTX2會與TRAF6結合但不會與NEMO或TAK1結合 18
8. DTX2抑制TRAF6所主導的κB啟動子活化 18
9. DTX2不影響TRAF6表現量 18
10. DTX2缺失陷增強TLR活化小鼠腹腔巨噬細胞引發的TRAF6/NEMO/IKKβ複合體形成 19
11. DTX2透過Ring-Finger及proline-rich 區段與TRAF6 進行結合
20
12. DTX2缺陷增加小鼠敗血性休克死亡 20
討論 21
參考資料 26
圖表 31

圖表目錄
圖一、缺失DTX2會增強小鼠骨髓巨噬細胞於TLR活化下之TNF-α產生。 31
圖二、缺失DTX2會增強小鼠骨髓巨噬細胞於TLR活化下之IL-6的產生 32
圖三、缺失DTX2會增強小鼠骨髓巨噬細胞於TLR活化下之IL-1β產生 33
圖四、缺失DTX2會增加小鼠腹腔巨噬細胞於TLR活化下之IFN-α信使核醣核酸表現 34
圖五、缺失DTX2會增加小鼠腹腔巨噬細胞於TLR活化下之IFN-β信使核醣核酸表現 35
圖六、THP-1細胞中以shRNA下調DTX2之表現。 36
圖七、THP-1細胞下調DTX2會增強TLR活化下之TNF-α產生 37
圖八、THP-1細胞下調會DTX2增強TLR活化下之IL-6產生 38
圖九、DTX2之缺失會增強小鼠骨髓巨噬細胞於TLR活化下IKK/ΙκΒα磷酸化 39
圖十、DTX2缺失些微影響小鼠腹腔巨噬細胞於TLR活化下ERK/Akt磷酸化 40
圖十一、DTX2會和TRAF6結合 41
圖十二、DTX2不會和TAK1及NEMO結合 42
圖十三、DTX2不影響TRAF6表現量 43
圖十四、DTX2抑制TRAF6主導的κΒ啟動子活化 44
圖十五、缺失DTX2會增強小鼠腹腔巨噬細胞於TLR活化下之TRAF6/NEMO/IΚΚβ複合體結合 45
圖十六、不同截切段的DTX2表現質體 46
圖十七、DTX2透過Ring-Finger及Proling-rich 區段與TRAF6 進行結合 47
圖十八、DTX2缺陷增加小鼠敗血性休克死亡率 48
附錄一、TLR2刺激下引起的NF-κΒ活化 49
附錄二、DTX及哺乳類DTX家族成員結構 50

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