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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80405
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor林琬琬(Wan-Wan Lin)
dc.contributor.authorLing-Ya Chiuen
dc.contributor.author邱鈴雅zh_TW
dc.date.accessioned2022-11-24T03:05:57Z-
dc.date.available2022-02-16
dc.date.available2022-11-24T03:05:57Z-
dc.date.copyright2022-02-16
dc.date.issued2022
dc.date.submitted2022-02-05
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80405-
dc.description.abstractPoly(ADP-ribose) polymerase-1 (PARP-1)是廣為熟知的修復去氧核醣核酸(DNA)起始酵素,因為它可以催化二磷酸腺苷核糖的聚合反應,修飾並調控DNA修復相關的酵素。更多研究逐漸發現,PARP-1在許多疾病模式中會調控發炎反應,因此我們致力於研究PARP-1如何調控NLRP3發炎小體(inflammasome)活化以及紫外光B誘發之發炎反應。位於胞內的核苷酸結合寡聚化結構域樣受體NLRP3,由於它可以偵測到廣泛的病原體入侵以及源自於細胞損傷釋出的危險訊號因子,進而啟動下游的免疫反應,是很重要的先天免疫反應受體。我們的研究發現,在小鼠骨髓分化巨噬細胞中,NLRP3刺激劑誘發的caspase-1活化及白介素-1貝他(IL-1)釋放,都受到PARP-1基因缺損或是PARP-1抑制劑所抑制。然而,活化NLRP3發炎小體的第一步訊息並未受到PARP-1基因缺失影響。除此之外,三磷酸腺苷(ATP)誘發之胞內氧化自由基(ROS),在PARP-1缺損之小鼠骨髓誘發巨噬細胞產生的較少,因而導致發炎小體複合體聚集較少。PARP-1會在ATP的刺激下從細胞核內轉移到細胞質,去催化NLRP3的聚(ADP-核糖基)化(PARylation)修飾,進而促進NLRP3發炎小體複合體的組成。PARP-1同時也做為NLRP3和TXNIP蛋白的橋樑,並參與在NLRP3/TXNIP複合體的生成,因此促進發炎小體的活化。我們的研究找到PARP-1調控NLRP3發炎小體活化的嶄新機制,因此PARP-1有潛力成為發展治療IL-1相關疾病的藥物標的。 紫外光會損傷皮膚,造成曬傷、發炎以及相關的皮膚疾病,在第二部分的研究中,我們想了解PARP-1如何調控紫外光B造成的皮膚損傷以及和表皮生長因子受體(EGFR)間的交互作用和其相關的發炎反應。我們發現PARP-1的基因缺損會惡化小鼠對於紫外光B誘發之發炎反應、水分散失以及背部皮膚損傷。在人類原發性角質細胞中,紫外光B會活化PARP-1並在PARP-1基因默化時造成更嚴重的DNA損傷。而紫外光B誘發之環氧合酶-2(COX-2)和基質金屬蛋白酶-1(MMP-1)的表現,會受到PARP-1基因靜默或其抑制劑所抑制,然而,腫瘤壞死因子阿法(TNF-和白介素-8 (IL-8)的生成卻因此而增強。由於EGFR基因靜默或是用其抑制劑gefitinib會抑制紫外光B誘發之COX-2、TNF-以及IL-8的表現量,顯示EGFR經由旁分泌的活化,會進而影響紫外光B誘發之發炎反應。西方墨點法的結果顯示,抑制PARP-1會降低紫外光B引發之EGFR和p38 MAPK活化,而p38 MAPK抑制劑會明顯抑制紫外光B誘發之發炎反應。值得注意的是,PARP-1或是EGFR的基因靜默都會降低紫外光B誘發之ROS生成,而抗氧化劑NAC會降低紫外光B引發的EGFR-p38 MAPK訊息傳遞和PARP-1活化。以上結果顯示,在EGFR、PARP-1和ROS之間有相互調控的迴路。因此,紫外光B的刺激下,PARP-1不只是參與DNA修復的功能,同時也會經由EGFR活化及ROS生成間的交互作用,造成下游p38 MAPK的訊息傳遞,進而調控角質細胞的發炎反應。zh_TW
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dc.description.tableofcontents"口試委員會審定書…………………………………………………………i 誌謝………………………………………………………………………...ii 中文摘要…………………………………………………………………..iii 英文摘要…………………………………………………………………...v ABBREVIATIONS……………………………………………………..viii INTRODUCTION………………………………………………………...1 1. Poly(ADP-ribose) polymerase-1……………………………………………….1 1.1 PARP-1 and ADP-ribosyltransferase………………………………………1 1.2 The domain structure of PARP-1…………………………………………...2 1.3 The function of PARP-1 in DNA repair and as therapeutic target in cancer..3 1.4 The relationship between PARP-1 and inflammation………………………...4 2. NOD-like receptor (NLR) inflammasome……………………………………...6 2.1 The NLRs family…………………………………………………………….6 2.2 The NLRP3 inflammasome………………………………………………..7 2.3 PARP-1 and NLRP3 inflammasome……………………………………….9 3. UVB-induced inflammation in skin…………………………………………...10 SPECIFIC AIMS………………………………………………………...13 MATERIALS AND METHODS………………………………………...15 1. Reagents and antibodies……………………………………………………….15 2. Mice and in vivo experiments…………………………………………………17 3. Immunohistochemical staining………………………………………………..18 4. Cell culture…………………………………………………………………….18 5. Candida albicans infection……………………………………………………19 6. UVB irradiation……………………………………………………………….20 7. Enzyme-linked immunosorbent assay (ELISA)……………………………….20 8. Immunoblot analysis…………………………………………………………..20 9. Immunoprecipitation………………………………………………………….21 10. Quantitative real-time RT-PCR analysis (QPCR) ……………………………..22 11. Plasmids and transient transfections…………………………………………...24 12. Small interference RNA (siRNA) transfection………………………………...24 13. ASC cross-linking assay……………………………………………………....25 14. Cytosolic and mitochondrial ROS detection…………………………………..25 15. Nuclear fractionation………………………………………………………….26 16. Immunofluorescence and confocal microscopy……………………………….27 17. Poly(ADP-ribosyl)ation in vitro……………………………………………….27 18. Annexin V/PI staining and flow cytometry……………………………………28 19. MTT assay…………………………………………………………………….28 20. Lactate dehydrogenase (LDH) assay…………………………………………..29 21. Statistical analysis……………………………………………………………..29 RESULTS……………………………………………………………………………...30 Part I: To understand the role of PARP-1 in NLRP3 inflammasome activation in macrophages…………………………………………………………………………...30 1. PARP-1 knockout decreases various stimuli-induced NLRP3 activation…....30 2. PARP-1 deficiency attenuates caspase-1 activation and decreases NLRP3 inflammasome assembly with no effect on NLRP3 complex expression….....31 3. PARP activator and inhibitor oppositely regulate NLRP3 activation………...32 4. PARP-1 deficiency decreases cytosolic and mitochondrial ROS production...33 5. ROS induce cytosolic translocation of PARP-1 and the interaction of PARP-1 with TXNIP and NLRP3………………………………………………….......34 6. PARP-1 can interact with TXNIP and NLRP3 and mediate NLRP3 PARylation.......................................................................................................36 7. PARP-1 deficiency decreases LPS-triggered peritonitis…………………......37 Part II: To understand the role of PARP-1 in UVB-induced inflammation in mice skin and keratinocytes………………………………………………………………...39 1. PARP-1 deficiency exacerbated the UVB-induced skin lesions in vivo………39 2. PARP-1 mediates UVB-induced DNA repair in keratinocytes………………39 3. PARP-1 differentially regulates UVB-induced COX-2, MMP-1, TNF- and IL-8 expression in keratinocytes………………………………………………....41 4. EGFR is involved in inflammation response to UVB………………………..42 5. PARP-1 contributes to EGFR/p38 MAPK signaling axis for UVB-induced inflammation…………………………………………………………………43 6. PARP-1 positively regulates UVB-induced ROS regulation and subsequent EGFR activation………………………………………………………….......44 DISCUSSION………………………………………………………………………….47 CONCLUSION………………………………………………………………………..61 REFERENCES………………………………………………………………………..63 FIGURES AND LEGENDS…………………………………………………………..84 APPENDIX…………………………………………………………………………...110 "
dc.language.isoen
dc.subject角質細胞zh_TW
dc.subject發炎小體zh_TW
dc.subject發炎反應zh_TW
dc.subject巨噬細胞zh_TW
dc.subject紫外光Bzh_TW
dc.subjectEGFRen
dc.subjectPARP-1en
dc.subjectNLRP3en
dc.subjectinflammasomeen
dc.subjectUVBen
dc.subjectinflammationen
dc.titlePoly(ADP-ribose) Polymerase-1調控巨噬細胞NLRP3發炎小體活化及紫外光B誘發角質細胞發炎反應之角色zh_TW
dc.titleThe Roles of Poly(ADP-ribose) Polymerase-1 in Regulation of NLRP3 Inflammasome Activation in Macrophages and UVB-induced Inflammation in Keratinocytesen
dc.date.schoolyear110-1
dc.description.degree博士
dc.contributor.oralexamcommittee洪啓峯(Wen-I Chuang),吳南霖(Daniel Wei-Chung Miao),徐立中,蔡丰喬
dc.subject.keyword發炎小體,發炎反應,巨噬細胞,紫外光B,角質細胞,zh_TW
dc.subject.keywordPARP-1,NLRP3,inflammasome,UVB,inflammation,EGFR,en
dc.relation.page116
dc.identifier.doi10.6342/NTU202200036
dc.rights.note同意授權(限校園內公開)
dc.date.accepted2022-02-09
dc.contributor.author-college醫學院zh_TW
dc.contributor.author-dept藥理學研究所zh_TW
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