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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 徐立中(Li-Chung Hsu) | |
dc.contributor.author | Miao-Hsien Wu | en |
dc.contributor.author | 吳苗嫻 | zh_TW |
dc.date.accessioned | 2021-06-08T01:44:54Z | - |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-15 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19095 | - |
dc.description.abstract | 先天性免疫系統是宿主抵禦外來病原體入侵和體內細胞受損的第一道防線,藉由體內多種的pattern recognition receptor (PRRs) 的活化而啟動。Toll-like receptors (TLRs) 屬於 PRRs的一種,可辨識來自病原體pathogen associated-molecular patterns (PAMPs)以及來自體內受損細胞的damage associated-molecular patterns (DAMPs) ,並透過MyD88依賴性的訊息傳導途徑與TRIF依賴性的訊息傳導途徑誘發發炎細胞激素和第一型干擾素的表現,進而引發發炎反應等一連串先天性免疫反應。然而,失衡且無法控制的免疫反應將對宿主造成嚴重的傷害,因此精細而縝密的調控對TLR極為重要。我們實驗室之前發現了一個新穎的鋅指蛋白 ―ZFAND5,其在巨噬細胞內表現量會在脂多醣(LPS)的刺激下明顯增加。ZFAND5是個23kDa大小的細胞質蛋白,其N端含有一個類似A20的鋅指結構,並在C端含有一個類似AN1的鋅指結構。雖然過去ZFAND5曾被報導過會與TLR訊息傳導途徑中的IKKγ, RIP, TRAF6以及polyubiquitin chain互相作用,但是其在TLR中所扮演的角色仍是不清楚的。
在本篇研究中,我們將研究的焦點集中於ZFAND5在TLR4所引導的免疫反應中的作用與其分子機制。我們首先利用CRISPR/Cas9的系統生產出完全剔除ZFAND5的巨噬細胞,並確認了其IRF3與第二波IKK的活化與先前藉由shRNA降低ZFAND5表現量的巨噬細胞相同,在ZFAND5缺失的狀況下都有所減少。此外,我們也揭露了ZFAND5會與TRAF3結合,並藉由促進TRAF3和TRIF的交互作用,增強TRAF3的K63-linked polyubiquitination。綜合以上結果,我們的結果顯示著ZFAND5在TLR4所引發的發炎反應中扮演一個正向調控的角色 | zh_TW |
dc.description.abstract | Innate immunity is the first-line defense to protect the host from pathogenic infection and cellular damage. It is initiated by various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). TLRs can sense pathogen associated-molecular patterns (PAMPs) derived from microbes and damage associated-molecular patterns (DAMPs) from stressed/damaged cells, and then induce the production of proinflammatory cytokines and type I interferon. Uncontrolled inflammatory response is harmful to the host. Therefore, the precise regulation of TLRs-triggered innate immune response is important. We previous identified a novel zinc finger protein, ZFAND5, which is up-regulated in LPS stimulated-macrophages. ZFAND5 is a 23-kDa cytosolic protein containing an A20-like zinc finger domain at its N terminus and an AN1-like domain at its C terminus. It has been reported that ZFAND5 interacts with several proteins, such as IKKγ, RIP, TRAF6 and polyubiquitin, which are all involved in TLR-mediated immune responses. However, the role of ZFAND5 in regulation of TLRs signaling is still unclear. Here, we focus on the function of ZFAND5 in TLR4-driven immune response and its molecular mechanism. We first generate ZFAND5-/- RAW264.7 cells using the CRISPR/Cas9 system. Similar to ZFAND5 knock-down cells mediated by shRNA, we confirmed that activation of IRF3 and late-phase IKK was decreased in ZFAND5-/- macrophages. In addition, we reveal that ZFAND5 interacts with TARF3 and mediates its K63-linked polyubiquitination by promoting the interaction of TRIF and TRAF3. Together, our results suggest that ZFAND5 acts as a positive regulator in TLR4-triggered immune response. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:44:54Z (GMT). No. of bitstreams: 1 ntu-105-R03448001-1.pdf: 4120741 bytes, checksum: dfdb8f1721e1d9d52424594b5c52885c (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 中文摘要 III Abstract IV Introduction 1 Innate immunity 1 Toll-like receptors (TLRs) 2 TLRs signaling pathways 3 TIR-domain-containing adaptors 3 The formation of Myddosome and the MyD88-dependent pathway 4 The formation of Triffosome and the TRIF-dependent pathway 6 TLR4 signaling pathway 7 Ubiquitination in TLR-mediated signaling pathways 7 Zinc-finger containing proteins 9 TLR-inducible Zinc-finger containing proteins 10 ZFAND5 10 Specific Aim 12 Materials and Methods 13 Reagents 13 Antibodies 13 Plasmids 14 Purification of His-tagged ZFAND5 antigen 15 Generation of anti-ZFAND5 antiserum 16 Cell culture 17 Transfection 17 Generation of ZFAND5-/- RAW264.7 macrophages 18 Preparation of whole cell lysates 19 Immunoblotting 19 Immunoprecipitation 20 Total RNA extraction 21 Reverse Transcription Quantitative PCR (RT-qPCR) 21 Enzyme-linked immunosorbent assay (ELISA) 22 Statistical analysis 23 Results 24 Generation of anti-ZFAND5 antiserum 24 Generation of ZFAND5-/- RAW264.7 cells using CRISPR/Cas9 system 25 Depletion of ZFAND5 in RAW264.7 macrophages decreases TLR4-induced activation of IRF3 and late phase IKK 26 ZFAND5 depletion in RAW264.7 macrophages decreases activation of IKK and IRF3 in response to poly(I:C) 27 ZFAND5 regulated K63-linked ubiquitination of TRAF3 in RAW264.7 macrophages after LPS stimulation 27 ZFAND5 depletion suppresses the interaction of TRIF and ZFAND5 28 Discussion 30 Figures 35 Figure 1 36 Figure 2 37 Figure 3 40 Figure 4 42 Figure 5 45 Figure 6 46 Figure 7 47 Figure 8 48 Supplementary Figure 1 49 Supplementary Figure 2 51 Appendix 52 Appendix 1. TLR4 signaling pathway 52 References 54 | |
dc.language.iso | en | |
dc.title | 一個新穎的鋅指蛋白於TLR4引導的免疫反應中所扮演的角色 | zh_TW |
dc.title | The role of a novel zinc finger protein in TLR4-mediated immune responses | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 盧主欽(Juu-Chin Lu),胡孟君(Meng-Chun Hu) | |
dc.subject.keyword | 先天性免疫,鋅指蛋白, | zh_TW |
dc.subject.keyword | Innate immunity,TLR,zinc finger protein, | en |
dc.relation.page | 61 | |
dc.identifier.doi | 10.6342/NTU201602749 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-16 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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