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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李建國(Chien-Kuo Lee) | |
dc.contributor.author | Wei-Bei Wang | en |
dc.contributor.author | 王偉蓓 | zh_TW |
dc.date.accessioned | 2021-06-13T06:40:14Z | - |
dc.date.available | 2016-10-05 | |
dc.date.copyright | 2011-10-05 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-20 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35073 | - |
dc.description.abstract | 在先天性的免疫力(innate immunity)中的第一型干擾素(type I IFN)是一種用來對抗病毒入侵的強效細胞素(cytokine)。目前已知第一型干擾素的刺激會活化細胞中的三種訊號傳導與轉錄子(STAT)蛋白:分別是STAT1、STAT2和STAT3。利用基因剔除小鼠的方式已證實STAT1和STAT2在第一型干擾素引起的抗病毒反應扮演非常重要的正調控角色,但是STAT3的角色並不清楚。為了研究這個議題,我們利用STAT3基因剔除的小鼠胚胎纖維母細胞株(MEF cell line)和小鼠骨髓衍生的初代巨噬細胞(primary BMM)證實了第一型干擾素的訊號在STAT3基因剔除的狀況下會增強。經由微陣列分析(microarray)和反轉錄即時聚合酶鏈式反應(RT-QPCR)的分析結果,我們發現第一型干擾素下游的抗病毒基因的表現量在STAT3基因剔除的小鼠細胞中比對照組的細胞高。我們也進一步地利用腦心肌炎病毒(EMCV)或疱疹性口腔炎病毒(VSV)直接感染細胞。實驗結果發現,STAT3基因剔除的小鼠細胞表現出較強的抗病毒能力,因此存活率比對照組細胞高且病毒效價(viral titer)比對照組細胞低。在將具有正常功能的STAT3回復到STAT3基因剔除的小鼠肧胎纖維母細胞株或利用介白素6 (IL-6) 的刺激來高度活化細胞中的STAT3,我們發現STAT3會抑制第一型干擾素的訊號以及其引起的抗病毒反應。證實了STAT3活性的增加的確可以負調控第一型干擾素的訊號和抗病毒反應。令人驚訝的是,我們發現不具有轉錄能力的STAT3蛋白質N端1-134 a.a. 就足以具有抑制效果。我們同時也發現了無論是腦心肌病毒的感染或是類鐸受體作用劑(TLR agonist)的刺激下,STAT3基因剔除的小鼠細胞會產生比較高量的第一型干擾素。我們進一步利用報導基因表現分析(reporter assay)證實了STAT3可能具有抑制聚肌胞苷酸 (poly (I:C))或干擾素轉接蛋白(MAVS)下游的第一型干擾素基因表現能力。以上的研究結果證實了STAT3負調控抗病毒反應的作用機制可能有兩種。第一,STAT3可以直接負調控第一型干擾素的訊息傳導以及其誘發的抗病毒能力。第二,STAT3也具有負調控第一型干擾素基因表現的能力,因而逹到抑制抗病毒反應的效果。以上的實驗結果證實了STAT3在第一型干擾素或是Toll樣受體所誘發的抗病毒反應中扮演著負調控的角色。 | zh_TW |
dc.description.abstract | Type I interferons (IFNs) are potent cytokines for innate immunity to combat viral infections. It has been well documented that signal transducer and activator of transcription (STAT) proteins such as STAT1, STAT2, and STAT3 are activated upon IFN-α/β stimulation. While essential roles of STAT1 and STAT2 in type I IFN-mediated antiviral responses are demonstrated in gene-targeting mice, the role of STAT3 remains unclear. Using STAT3KO mouse fibroblasts (MEFs) and primary bone-marrow-derived macrophages (BMMs) lacking STAT3, we demonstrated that IFN-α signals in STAT3KO MEFs or BMMs were enhanced. Both microarray and RT-QPCR analysis revealed that the induction of several IFN-a-inducible antiviral-associated genes was higher in STAT3KO cells than that in wild-type cells in response to IFN-α. Moreover, STAT3KO cells also displayed increased antiviral responses to encephalomyocarditis virus (EMCV) or vesicular stomatitis virus (VSV) infections with increased survival levels and decreased viral titers. STAT3 restoration to STAT3KO MEFs or STAT3 hyper-activation by IL-6 stimulation attenuated the enhanced type I IFN signals and antiviral responses, suggesting that STAT3 negatively regulates these two responses. Surprisingly, STAT3 1-134 a.a. was sufficient to reverse the hyper-antiviral activity. We also found that EMCV infection or TLR agonists stimulation induces the increased level of IFN-α/β in the absence of STAT3 in vitro or in vivo. Additionally, STAT3 might suppress the IFN-β promoter activity upon poly (I:C) treatment and mitochondrial antiviral signaling (MAVS)-mediated signaling pathway. Therefore, at least, two mechanisms might contribute to the enhanced antiviral activity, namely enhanced signaling of type I IFNs and increased IFN-α/β production, in the cells lacking STAT3. Taken together, these results suggest an important and yet previously uncharacterized role of STAT3 in IFN-α/β and TLR-mediated antiviral responses. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:40:14Z (GMT). No. of bitstreams: 1 ntu-100-D93449002-1.pdf: 8192758 bytes, checksum: 244e963c03e7509995230f0b4017a05d (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Contents
口試委員會審定書 i Acknowledgements ii Abstract iv 中文摘要 vi Contents viii Index of figures X Index of tables XII 1. Introduction 1 1.1 Pattern-recognition receptors 1 1.1.1 Toll-like receptors 2 1.1.2 RIG-I-like receptors 5 1.1.3 NOD-like receptors 8 1.1.4 C-type lectin receptors 10 1.1.5 DNA sensors 12 1.2 Type I IFNs 14 1.2.1 Biological activities of type I IFNs 15 1.2.2 Regulation of type I IFN production 16 1.2.3 Effects of type I IFNs 18 1.2.4 Type I IFN signaling pathway 21 1.3 The molecules in type I IFN signaling 23 1.3.1 The JAKs in type I IFN signaling 23 1.3.2 The STATs in type I IFN signaling 24 1.3.3 Negative regulation of JAK-STAT pathway 26 1.4 STAT3 28 1.4.1 Biological implications of STAT3 28 1.4.2 Phenotypes of tissue-specific knockout of STAT3 30 2. Aim of the thesis 36 3. Materials and methods 38 3.1 Materials 38 3.1.1 Chemicals and Reagents 38 3.1.2 Buffers, media and solutions 39 3.1.3 TLR ligands 41 3.1.4 Primer sequences 42 3.1.5 Recombinant cytokines 44 3.1.6 Antibodies 44 3.1.7 Cell lines 45 3.1.8 Mouse and virus strains 46 3.1.9 Plasmids 46 3.2 Methods 48 3.2.1 Induction of STAT3 deletion in mice 48 3.2.2 Virus propagation 48 3.2.3 In vitro differentiation of primary BMMs 49 3.2.4 RNA interference 49 3.2.5 Calcium phosphate precipitation transfection 50 3.2.6 TurbofectTM in vitro transfection 50 3.2.7 Retroviral transduction 51 3.2.8 RT-QPCR 51 3.2.9 Western blot analysis and co-immunoprecipitations 51 3.2.10 Plaque formation assay 52 3.2.11 In vitro antiviral assay 52 3.2.12 Propidium iodide (PI) staining 53 3.2.13 Microarray analysis 53 3.2.14 Reporter activity assay 53 3.2.15 Preparation of cytosolic and nuclear extracts 54 3.2.16 ELISA 54 3.2.17 Statistics 54 4. Results 55 4.1 Silencing of STAT3 results in enhanced IFN-α responses 55 4.2 STAT3 ablation results in enhanced IFN-α signals 56 4.3 STAT3 ablation results in enhanced antiviral states against virus infection 57 4.4 Restoration of STAT3 reverses the hyper-antiviral state in STAT3KO cells 59 4.5 Hyper-activation of STAT3 by IL-6 stimulation inhibits IFN-α-stimulated gene induction and antiviral responses 59 4.6 IL-6 triggers antiviral activity in cells lacking STAT3 61 4.7 STAT3 ablation leads to increase of IFN-α-mediated signals during virus infection 62 4.8 Increased production of type I IFNs in STAT3KO cells during viral infection 63 4.9 MDA5 knockdown impedes enhanced antiviral responses in STAT3 cells 64 4.10 STAT3 directly suppresses IFN-α-induced gene expression 65 4.11 DBD and TAD domains of STAT3 are not required for suppressive effect of STAT3 66 4.12 NTD of STAT3 is sufficient to suppress type I IFN responses 67 4.13 Negative regulation of IFN-α signals by STAT3 is independent of interrupting STAT1-STAT2 formation or nuclear translocation of pSTAT1 or pSTAT2 67 4.14 STAT3 may negatively regulate TLR-mediated response 69 4.15 IL-10-enhanced TLR7 responses in STAT3KO BMMs is independent of the loss of IL-10 immune suppressive effect 69 4.16 Increased production of type I IFNs in STAT3KO cells in responses to IFN-α 70 4.17 STAT3 suprresses Poly (I:C) , MAVS, or IRF3/7-mediated IFN-β transcription activity 71 5. Discussion 72 5.1 Molecular mechanism of STAT3-mediated inhibition of type I IFN responses 72 5.2 Opposing roles of STAT1 and STAT3 on biological responses 75 5.3 The suppressive role of STAT3 in TLR or RLR-mediated responses 78 5.4 The kinetics of STAT3 activation during viral infection 79 5.5 The clinical relevance of STAT3 in therapeutic application 80 5.6 Concluding remarks 81 References 82 Abbreviations 182 Appendix 194 | |
dc.language.iso | en | |
dc.title | STAT3在抗病毒反應中所扮演的角色的研究 | zh_TW |
dc.title | Role of STAT3 in Antiviral Responses | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 伍安怡(Betty Wu-Hsieh),林宜玲(Ling-Yi Lin),蔡錦華(Ching-Hua Tsai),廖經倫(Ching-Len Liao) | |
dc.subject.keyword | 干擾素,抗病毒, | zh_TW |
dc.subject.keyword | STAT3,IFN,Antiviral response, | en |
dc.relation.page | 194 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-21 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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