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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9968
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dc.contributor.advisor賴明宗(Ming-Zong Lai)
dc.contributor.authorI-Hsuan Chiangen
dc.contributor.author江逸萱zh_TW
dc.date.accessioned2021-05-20T20:52:37Z-
dc.date.available2016-10-05
dc.date.available2021-05-20T20:52:37Z-
dc.date.copyright2011-10-05
dc.date.issued2011
dc.date.submitted2011-08-05
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9968-
dc.description.abstractXIAP為抑制細胞凋亡蛋白家族的成員,有研究指出XIAP利用BIR1 domain和TAB1-TAK1結合,進而影響NF-κB的活化。然而在生理情況下各種配體活化NF-κB時,XIAP真正的角色一直沒有被證實。實驗室先前的研究發現Notch活化會抑制XIAP被泛素化及分解,使XIAP穩定存在。有其他的研究則是指出Notch會造成NF-κB活化。因此在本研究中,我們探討XIAP是否參與在TNFα,TCR及Notch訊息造成的NF-κB活化。我們產製了XIAP調降及XIAP過度表現之DO11.10 T細胞株。在以TNFα刺激XIAP調降或XIAP過度表現之DO11.10,結果顯示XIAP的調降或過度表現不影響TNFα刺激引起IκBα的磷酸化與分解以及p65進核。關於XIAP在TCR活化NF-κB的角色方面,下調XIAP的DO11.10細胞中,TCR引發的 IKK和IκBα的磷酸化減弱,p65進核部份抑制,κB啟動子的活性也降低。在過度表現的DO11.10的細胞中則觀察到相反的現象。但是PKCθ的磷酸化和Bcl10的表現量皆不受XIAP調降或過度表現影響。在研究XIAP於Notch活化NF-κB上的角色方面,我們以Notch配體Jagged-1刺激下調XIAP的DO11.10,實驗結果發現p65進核的量減少,Notch活化後NICD的生成也降低,但不影響Notch在細胞膜上的表現。zh_TW
dc.description.abstractX-linked inhibitor of apoptosis protein (XIAP) is a member of inhibitor of apoptosis protein (IAP) family. Previous studies reported that XIAP activates NF-κB by association with TAK1 which phosphorylates and activates IKK. The regulation of XIAP in stimulation through various physiological ligands for the activation of NF-κB, however, has not been confirmed. Previous study from our lab found that Notch intracellular domain (NICD) inhibits ubiquitination and degradation of XIAP. Other studies indicated that Notch can activate NF-κB. The specific aims of this study are to determine the role of XIAP in NF-κB activation induced by TNFα, T cell receptor (TCR) and Notch. We first established XIAP-knockdown and XIAP-overexpression DO11.10 T cell line. Upon stimulation with TNFα, the phosphorylation and degradation of IκBα, as well as p65 nucleus translocation, were not affected by knockdown and overexpression of XIAP. For the role of XIAP in TCR-induced NF-κB activation, XIAP deficiency reduced TCR-mediated phosphorylation of IKK and IκBα, p65 nucleus translocation, and κB promoter activation. Consistent with these results, XIAP overexpression increased the activation of NF-κB triggered by TCR. However, the activation of PKCθ and protein levels of Bcl10. But the phosphorylation of PKCθ and expression of Bcl10 proteins were not affected by XIAP knockdown or expression in TCR-stimulated DO11.10 T cells. For the role of XIAP in Notch-activated NF- κB, we found that knockdown of XIAP lead to decreased p65 nucleus translocation after Notch ligand stimulation. In addition, NICD formation and transcription of Notch target gene, dtx1, were also decreased.en
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dc.description.tableofcontents致謝 i
中文摘要 ii
英文摘要 iii
目錄 iv
圖目錄 vii
第一章 簡介
1.1 NF-κB 1
1.1.1 NF-κB 1
1.1.2 T細胞的活化與T細胞受體媒介的NF-κB活化 2
1.1.3 TNFα媒介的NF-κB活化 3
1.1.4 Notch媒介的NF-κB活化 3
1.2 XIAP 4
1.2.1 XIAP 4
1.2.2 XIAP的結構 4
1.2.3 XIAP的功能 6
第二章 材料與實驗方法
2.1 細胞株與細胞培養 8
2.1.1 細胞株 8
2.1.2 細胞培養 8
2.2 抗體 8
2.3 質體構築 8
2.3.1 pLL3.7-shXIAP 8
2.3.2 pcDNA.4-mXIAP-myc 9
2.3.3 pGC-YFP-mXIAP-myc 9
2.4 Lentivirus與Retrovirus反轉錄病毒的製備、效價測試與感染 10
2.4.1 Lentivirus反轉錄病毒的製備 10
2.4.2 Lentivirus反轉錄病毒的效價測試 10
2.4.3 Lentivirus反轉錄病毒感染 11
2.4.4 Retrovirus反轉錄病毒的製備 11
2.4.5 Retrovirus反轉錄病毒的感染 11
2.5 DNA的轉染 11
2.5.1 電穿孔法(Electroporation) 11
2.5.2 磷酸鈣轉染法(Calcium phosphate transfection) 12
2.6 T細胞活化 12
2.7 TNFα刺激 12
2.8 細胞質與細胞核萃取液的置備 12
2.9 冷光酵素活性測定試驗 13
2.10 西方墨點法分析 13
2.11 RNA萃取 14
2.12 反轉錄聚合酵素鏈鎖反應 14
第三章 結果 16
3.1 建立下調及過度表現XIAP的DO11.10細胞株 16
3.2 下調或過度表現XIAP不影響IAP家族cIAP1及cIAP2於細胞的
表現 16
3.3 下調XIAP減弱TCR引起的NF-κB活化 17
3.3.1 下調XIAP減弱TCR引起的IKK及IκBα磷酸化 17
3.3.2 下調XIAP減弱TCR引起的p65進核 17
3.3.3 下調XIAP減弱κB啟動子的活化 17
3.3.4 下調XIAP不影響TCR活化後PKCθ的磷酸化 18
3.3.5 下調XIAP不影響TCR活化後Bcl10的表現 18
3.4 過度表現XIAP增強TCR引起的NF-κB活化 18
3.4.1 過度表現XIAP增強TCR引起的IKK及IκBα磷酸化 18
3.4.2 過度表現XIAP增強κB啟動子的活化 19
3.4.3 過度表現XIAP不影響TCR活化後PKCθ的磷酸化 19
3.4.4 過度表現XIAP不影響TCR活化後Bcl10的表現 19
3.5 XIAP不影響TNFα引起的NF-κB活化 20
3.5.1 下調XIAP不影響TNFα引起的NF-κB活化 20
3.5.2 過度表現XIAP不影響TNFα引起的NF-κB活化 20
3.6 XIAP影響Notch引起的NF-κB活化 20
3.6.1 XIAP下調減少 Notch引起NF-κB p65的入核 21
3.6.2 XIAP影響Notch活化NICD的產生 21
3.6.3 XIAP影響Notch目標基因的轉錄 21
3.6.4 XIAP不影響Notch1受體於細胞膜上的表現 21
第四章 討論 22
圖表 26
參考文獻 44
附錄 52
dc.language.isozh-TW
dc.titleXIAP於活化NFkappaB訊息傳遞中扮演的角色zh_TW
dc.titleRole of XIAP in NFkappaB activationen
dc.typeThesis
dc.date.schoolyear99-2
dc.description.degree碩士
dc.contributor.oralexamcommittee繆希椿,李秀香
dc.subject.keywordX-linked 抗細胞凋亡蛋白,NFkappaB轉錄因子的活化,zh_TW
dc.subject.keywordXIAP,NFkappa activation,en
dc.relation.page57
dc.rights.note同意授權(全球公開)
dc.date.accepted2011-08-05
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