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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 | 蔡欣祐(Hsin-Yue Tsai) | |
dc.contributor.author | Kuan-Lin Ho | en |
dc.contributor.author | 何冠霖 | zh_TW |
dc.date.accessioned | 2021-06-08T01:10:13Z | - |
dc.date.copyright | 2020-08-26 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18532 | - |
dc.description.abstract | MCPIP1是一種核糖核酸酶,它能辨認轉譯中的信使核糖核酸上特殊莖環結構,並進一步將此信使核糖核酸剪切。MCPIP1並能透過這種方式調控一些細胞激素之信使核糖核酸的穩定性,像是IL-6。除此之外,MCPIP1也被發現能夠藉由剪切小分子核糖核酸前導體(pre-miRNAs)末端的環狀結構,進而抑制小分子核糖核酸(miRNAs)的生合成。另外,巨噬細胞(macrophage)的極化作用(polarization)對於巨噬細胞調控發炎以及對抗病原體是非常重要的。而分化後的巨噬細胞可以簡單的歸類為兩種類別:典型活化巨噬細胞(classical activated macrophage)和另類活化巨噬細胞(alternatively activated macrophage),這兩種巨噬細胞分別具有促進發炎與抑制發炎的能力。先前的研究結果發現MCPIP1在白血球介素-4(IL-4)刺激巨噬細胞分化成另類活化巨噬細胞的過程中扮演關鍵作用因子。然而,在骨髓源性巨噬細胞(bone marrow-derived macrophages)中,內源性MCPIP1蛋白的表現位置以及是否有其它相互作用蛋白(protein interactor)和MCPIP1一起作用都是未知的。我們的研究目的是找出骨髓源性巨噬細胞中,內源性MCPIP1蛋白的表現位置以及和其作用的新穎交互作用蛋白。本篇我們運用蛋白質體學分析方式找出一些在骨髓源性巨噬細胞中,可能和內源性MCPIP1相互作用的蛋白質。我們進而透過免疫共沉澱法篩選出兩種蛋白質:AGO2和DHX30,AGO2參與RNA干擾(RNAi)機制及具有和小分子核糖核酸(miRNAs)結合並調控對應到的信使核醣核酸的穩定性及產生蛋白的能力,而DHX30為一種RNA解旋酶並在先前被發現會和AGO2產生交互作用。我們還發現MCPIP1與AGO2及DHX30的交互作用並不需要依賴RNA。另外,我們也利用亞細胞分離法與免疫螢光染色的方式發現在骨髓源性巨噬細胞中,絕大多數的內源性MCPIP1蛋白表現在細胞質,但在細胞核中也具有少數的內源性MCPIP1蛋白表現。我們的研究結果提供了在骨髓源性巨噬細胞中,新發現的內源性MCPIP1之交互作用蛋白,並開拓探討MCPIP1與AGO2在巨噬細胞中之相互作用機制此一新穎研究方向。 | zh_TW |
dc.description.abstract | Monocyte chemotactic protein-induced protein 1 (MCPIP1, also known as Zc3h12a or Regnase-1), was found as ribonuclease which can cleave actively translated mRNAs with a specific recognition motif. MCPIP1 had also been shown regulating the mRNA turnover of several cytokines, such as IL6. Moreover, MCPIP1 also has been found inhibiting miRNA biosynthesis by cleaving the terminal loops of precursor miRNAs (pre-miRNAs). Macrophage polarization is critical for inflammation and pathogen response. The polarity of macrophage can be roughly classified into two subtypes, classical activated (pro-inflammatory) and alternatively activated (anti-inflammatory) macrophage. A previous study has shown that MCPIP1 is critical in IL-4 dependent alternatively activated macrophage polarization. However, where endogenous MCPIP1 is localized during macrophage polarization and what kinds of the protein interact with endogenous MCPIP1 in bone marrow-derived macrophages (BMDM) are still unknown. The purpose of our study is to find out the novel protein interactors and the subcellular localization of endogenous MCPIP1 in BMDM.To reveal endogenous MCPIP1 protein-interactors in BMDM, we used co-immunoprecipitation and proteomic analysis. We found interesting protein interactors of MCPIP1, such as AGO2 which is important in RNA interference (RNAi) and microRNA (miRNA) mediated silencing, and DHX30 which is RNA helicase and an interactor of AGO2. We further demonstrated that the interaction of MCPIP1 with AGO2 and DHX30 is RNA independent. Moreover, we used subcellular fractionation and immunofluorescence to discover the localization of endogenous MCPIP1 in BMDM. We found most MCPIP1 protein expressed in cytoplasm, but a little amount of MCPIP1 protein is also found in nucleus. Our findings open the possibility that MCPIP1 may work with AGO2 to regulate mRNA stability in macrophages. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:10:13Z (GMT). No. of bitstreams: 1 U0001-1208202011045300.pdf: 3182563 bytes, checksum: 96b6ed5fe5b6615d7f105c09471cf2be (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 口試委員會審定書 i 謝辭 ii 摘要 iii Abstract v Table of Contents vii Chapter 1 INTRODUCTION 1 1.1 Description of macrophage 1 1.2 MCPIP1- a regulator in the polarization of macrophages 3 Chapter 2 MATERIALS AND METHODS 6 2.1 Cell isolation and culture conditions 6 2.2 IL-4 induction of BMDM 6 2.3 Subcellular fractionation 7 2.4 Protein extraction 8 2.5 Immunoprecipitation 8 2.6 Western blot analysis 9 2.7 Proteomic analysis 10 2.8 Immunofluorescence staining 11 Chapter 3 RESULTS 13 3.1 Protein expression level of endogenous MCPIP1 in different mouse organs 13 3.2 Screening the potential protein interactors of MCPIP1 in mouse liver and spleen 14 3.3 Searching the potential protein interactors of MCPIP1 in BMDM with and without IL-4 induction 18 3.4 Checking the candidates of MCPIP1 interactors by co-immunoprecipitation 21 3.5 The protein-protein interaction of MCPIP1 with AGO2 and DHX30 is independent of RNA 23 3.6 MCPIP1 localizes in the nucleus and the cytoplasm of BMDM 24 Chapter 4 DISCUSSION 27 4.1 The difference of endogenous MCPIP1 protein expression levels in mouse organs between our data and previous study and between different MCPIP1 antibodies 27 4.2 The protein interactor of MCPIP1- AGO2 is involved in RNA-mediated gene silencing pathway 28 4.3 Endogenous MCPIP1 protein expresses in both of cytoplasm and nuclear of BMDM 31 Chapter 5 FIGURES 32 Figure 1. The expression of MCPIP1 protein in mouse organs 32 Figure 2. Potential protein interactors of MCPIP1 in mouse liver and spleen 35 Figure 3. Screening of MCPIP1 protein interactors in BMDM 40 Figure 4. The co-immunoprecipitation of endogenous MCPIP1 and the candidates of MCPIP1 protein interactors 43 Figure 5. The interaction of MCPIP1 with AGO2 and DHX30 was independent of RNA 44 Figure 6. The subcellular localization of endogenous MCPIP1 in BMDM 47 References 48 Appendix 54 | |
dc.language.iso | en | |
dc.title | 鑑別骨髓源性巨噬細胞中內源性MCPIP1之新穎交互作用蛋白 | zh_TW |
dc.title | Identification of novel protein interactors of endogenous MCPIP1 in bone marrow-derived macrophages | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 許書豪(Shu-Hao Hsu),許弘明(Hong-Ming Hsu) | |
dc.subject.keyword | MCPIP1,AGO2,骨髓源性巨噬細胞,巨噬細胞極化作用,交互作用蛋白, | zh_TW |
dc.subject.keyword | MCPIP1,AGO2,BMDM,macrophage polarization,protein interactor, | en |
dc.relation.page | 72 | |
dc.identifier.doi | 10.6342/NTU202003059 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2020-08-14 | |
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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