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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡欣祐(Hsin-Yue Tsai) | |
| dc.contributor.author | Wun-Ting Wu | en |
| dc.contributor.author | 吳文婷 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:11:52Z | - |
| dc.date.copyright | 2022-10-17 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-25 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84446 | - |
| dc.description.abstract | 先天性免疫系統是保護宿主免於病源入侵的一道防線，主要參與在此的免疫細胞為巨噬細胞。巨噬細胞上具有許多toll-like receptors (TLRs)可以辨認不同的外源因子。其中TLR4是TLRs中最廣泛被研究的，當LPS接觸到受體便會激發下游訊號途徑。MCPIP1又名Regnase-1，是一核糖核酸酶，在LPS刺激的巨噬細胞中可以降解特定的前發炎激素，例如:IL-6,IL-1β或IL-12b，以避免過度的發炎反應發生。然而，MCPIP1如何辨認特定目標仍未知。為了瞭解MCPIP1如何找到特定目標，我們首先找尋MCPIP1的交互蛋白，透過免疫沉澱及質譜儀找到一RNA解旋酶---DHX30。 DHX30已被發現參與在粒線體核糖體的生合成、抵禦病毒及抑制轉譯。為了找尋DHX30在MCPIP1介導的發炎巨噬細胞中所扮演的角色，我們確認了MCPIP1及DHX30的交互作用，並發現了調降DHX30會使MCPIP1 mRNA及蛋白的表現量下降也降低了發炎激素的mRNA表現。此外，我們也發現調降DHX30抑制粒線體基因表現。我們的結果顯示了DHX30在巨噬細胞中與MCPIP1之間的交互作用以及在發炎反應激素調控中的潛在角色。 | zh_TW |
| dc.description.abstract | Innate immunity is important in the first line defense of against pathogenic invasion and the macrophage is the key immune cell that governs this process. Different pathogen-associated molecules are recognized by various Toll-like receptors (TLRs) that reside on the macrophage. TLR4, triggered by LPS, is the most well-characterized macrophage pro-inflammatory pathway. MCPIP1, also known as Regnase-1, is a ribonuclease that specifically down-regulates a handful of cytokines, IL-6, IL-1β, and IL-12p40 (IL-12b), in macrophages upon LPS treatment. However, how MCPIP1 recognizes its targets are largely unknown. To know how MCPIP1 gains its substrate specificity, we aimed to find protein interactors of MCPIP1. Through MCPIP1 immunoprecipitation (IP) followed by mass spectrometry, we have identified an RNA helicase, DHX30. DHX30, an ATP-dependent RNA helicase, is previously shown essential for mitochondrial ribosome assembly, antiviral processes, and translation repression. To identify whether DHX30 also play role in MCPIP1 mediated cytokine mRNA turnover in macrophage upon LPS treatment, we confirm the interaction between DHX30 and MCPIP1 in macrophages. Furthermore, we revealed that knockdown DHX30 not only decreased MCPIP1 mRNA and protein levels but also reduced the mRNA level of pro-inflammatory cytokines in RAW 264.7 cells. In addition, we confirmed depleting DHX30 results in mitochondrial coding gene downregulation in macrophages. Our results establish the interaction between DHX30 and MCPIP1 and their potential roles in pro-inflammatory macrophage cells. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:11:52Z (GMT). No. of bitstreams: 1 U0001-2109202214292200.pdf: 6453640 bytes, checksum: 591bc56a6d4dacbe1de63c141aa8acec (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 謝辭 I 摘要 II Abstract III Table of contents V Chapter 1 Introduction 1 1.1 Pro-inflammatory macrophage 1 1.2 MCPIP1 regulates pro-inflammatory cytokines 2 1.3 DHX30 functions in cell 3 Chapter 2 Material and Methods 5 2.1 Cell culture conditions and LPS stimulation 5 2.2 shRNA and siRNA target to DHX30 6 2.3 RNA extraction and qRT-PCR 7 2.4 Protein extraction 8 2.5 Immunoprecipitation 8 2.6 Western blot 9 2.7 Immunofluorescence staining 10 2.8 statistical analysis 11 2.9 RNA-seq resource and analysis 11 Chapter 3 Results 12 3.1 The localization of DHX30 and MCPIP1 in macrophages 12 3.2 DHX30 interacted with MCPIP1 13 3.3 Depletion of DHX30 or MCPIP1 decreased each mRNA and protein level 13 3.4 LPS treatment increased the level of MCPIP1 and pro-inflammatory cytokines 14 3.5 Knockdown DHX30 decreased MCPIP1 and pro-inflammatory cytokines expression upon LPS treatment 15 3.6 Knockdown DHX30 decreased mitochondrial coding mRNA 16 Chapter 4 Discussion 19 1. Pro-inflammatory cytokines downregulated in MCPIP1 decreased macrophages 19 2. Depletion of DHX30 enhanced global translation in the previous report 20 3. DHX30 downregulated mitochondrial coding gene level 21 Chapter 5 Figures 22 Figure 1. The localization of DHX30 and MCPIP1 in macrophages 26 Figure 2. The interaction of DHX30 and MCPIP1 in macrophages 27 Figure 3. Knockdown DHX30 decreased MCPIP1 mRNA and protein levels 29 Figure 4. LPS treatment increased the level of MCPIP1 and pro-inflammatory cytokines and the accumulation of MCPIP1 in macrophages’ dendritic-like extension 33 Figure 5. Knockdown DHX30 decreased MCPIP1 and pro-inflammatory cytokines expression upon LPS treatment 39 Figure 6. Knockdown DHX30 decreased mitochondrial coding gene expression and down-regulate mitochondrial related signaling pathway 43 Figure 7. DHX30 and MCPIP1 complex regulate proinflammatory cytokines and mitochondrial mRNA in LPS treatment macrophages 44 Tables 45 Table-I Primers for RT-qPCR 45 References 47 | |
| dc.language.iso | en | |
| dc.subject | 前發炎反應激素 | zh_TW |
| dc.subject | MCPIP1 | zh_TW |
| dc.subject | DHX30 | zh_TW |
| dc.subject | 巨噬細胞 | zh_TW |
| dc.subject | LPS | zh_TW |
| dc.subject | 前發炎反應 | zh_TW |
| dc.subject | pro-inflammation | en |
| dc.subject | pro-inflammatory cytokines | en |
| dc.subject | MCPIP1 | en |
| dc.subject | DHX30 | en |
| dc.subject | macrophages | en |
| dc.subject | LPS | en |
| dc.title | DHX30在MCPIP1介導之發炎巨噬細胞中所扮演的角色 | zh_TW |
| dc.title | A role of DHX30 in MCPIP1-mediated function in pro-inflammatory macrophages | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 朱家瑩(Chia-Ying Chu),詹世鵬(Shih-Peng Chan),徐立中(Li-Chung Hsu) | |
| dc.subject.keyword | MCPIP1,DHX30,巨噬細胞,LPS,前發炎反應,前發炎反應激素, | zh_TW |
| dc.subject.keyword | MCPIP1,DHX30,macrophages,LPS,pro-inflammation,pro-inflammatory cytokines, | en |
| dc.relation.page | 52 | |
| dc.identifier.doi | 10.6342/NTU202203723 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-09-26 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| dc.date.embargo-lift | 2027-09-25 | - |
| 顯示於系所單位： | 分子醫學研究所 | |
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