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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 江皓森(Hao-Sen Chiang) | |
| dc.contributor.author | Ci-Yun Huang | en |
| dc.contributor.author | 黃頎勻 | zh_TW |
| dc.date.accessioned | 2021-06-08T02:38:14Z | - |
| dc.date.copyright | 2018-07-26 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-20 | |
| dc.identifier.citation | 1.McNab, F., et al., Type I interferons in infectious disease. Nat Rev Immunol, 2015. 15(2): p. 87-103.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19971 | - |
| dc.description.abstract | 第一型干擾素的免疫反應是哺乳動物細胞防禦病毒感染的主要機制。已知GEF-H1 (Guanine nucleotide exchange factor) 會影響第一型干擾素的生成，因此我們希望了解是否與GEF-H1相似的同源蛋白: ARHGEF1 (Rho guanine nucleotide exchange factor 1) 也可以調控第一型干擾素的生成。
我們利用ARHGEF1表現有缺陷的細胞株Huh-7以及B6小鼠做為實驗模組。 在感染仙台病毒或是使用對RIG-I Like Receptor (RLR)有專一性的配體來刺激野生型與ARHGEF1-/-的Huh-7細胞後，ARHGEF1有缺陷的組別在第一型干擾素基因IFNB1和其下游引發的基因MX1的表現量顯著高於野生型的組別。此外，我們同時使用對RLR具專一性的配體來刺激由Arhgef1+/-小鼠的骨髓所分化的巨噬細胞或直接對Arhgef1+/-小鼠注射對RLR具專一性的配體，也發現有相同的趨勢，Ifnb1基因與Ifn-β表現在Arhgef1有缺陷的組別表現量較野生型組別高。此外，我們也做了ARHGEF1的重建實驗，我們將可以表現ARHGEF1全長蛋白和ARHGEF1不同結構域 (Domain) 的突變型蛋白像是RGS (Regulator G protein signaling)和DH (Dbl homology)以及PH (Pleckstrin homology)的質體轉入ARHGEF1-/- Huh-7細胞中，並且利用對RLR具專一性的配體來刺激細胞，發現全長型或是突變型的ARHGEF1都可以負調控IFNB1的表現量。 有趣的是，我們更發現ARHGEF1-/-Huh-7細胞中油滴 (lipid droplets) 的數量和脂質調控基因SREBP2(sterol regulatory element-binding protein 2) 的表現量比野生型要少，但是經由抗體阻隔第一型干擾素受體的實驗中，發現油滴數量並不受到第一型干擾素影響。 總結我們在細胞和動物體內的實驗中都發現ARHGEF1具有負調控RLR路徑引發的第一型干擾素合成。至於ARHGEF1和脂質代謝的關係目前尚未釐清。 | zh_TW |
| dc.description.abstract | Mammalian cells express type I interferons (IFNs) that are important for innate immune defense against viral infection. Given that guanine nucleotide exchange factor GEF-H1 is the critical regulator of type I IFN expression, we sought to determine whether rho guanine nucleotide exchange factor 1 (ARHGEF1), a GEF-H1 homolog, is also able to control type I IFN expression and responses by utilizing ARHGEF1-deficient Huh7 cells and Arhgef1+/- mice. The expressions of IFNB1 and interferon-stimulated gene (ISG), MX1 were both significantly increased in RIG-I-Like Receptor (RLR) ligands-incubated or Sendai virus-infected ARHGEF1-/-Huh7 cells compared to wild-type (WT) Huh7 cells. We also observed a similar phenotype that Arhgef1+/- bone marrow-derived macrophages (BMDMs) expressed elevated amounts of Ifnb1 than WT BMDMs in response to high molecular weight (HMW) poly(I:C) stimulation. Furthermore, the production of Ifn-beta was profound enhanced in the serum of Arhgef1+/- mice administrated with HMW poly(I:C) intraperitoneally. Mechanistically, reconstitution of ARHGEF1 and different ARHGEF1 mutants into ARHGEF1-/- Huh7 cells further indicated that the negative regulation of IFNB1 expression in response to RLR stimulus was dependent on the regulator of G protein signaling (RGS), Dbl homology (DH), and Pleckstrin homology (PH) domains of ARHGEF1.
Surprisingly, while IFN-beta is known to alter cellular lipid metabolism, blocking of IFNα/β receptor signaling revealed that the decrease of lipid content and expression of sterol regulatory element-binding-protein 2 (SREBP2), a critical transcriptional factor for cholesterol synthesis, in ARHGEF1-/- Huh7 cells was not due to the elevated IFNβ expression. Taken together, our results suggest that ARHGEF1 negatively regulates RLR-mediated type I IFN expression both in vitro and in vivo and may have an additional role in the regulation of lipid metabolism. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T02:38:14Z (GMT). No. of bitstreams: 1 ntu-107-R05b21012-1.pdf: 3897631 bytes, checksum: d2a6e7cb4c308237e54836aaddbdddb0 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員會審定書........................................I
致謝...................................................II 中文摘要...............................................III Abstract...............................................V 目錄...................................................VII Chapter 1 Introduction................................ 1 1.1 Type one interferon (Type I IFN)............... 1 1.1.1 Antivirus response and interferon response...... 1 1.1.2 Type I IFN classification....................... 2 1.1.3 Type I IFN production and signaling............. 3 1.1.4 Type I interferon and GEF-H1................... 7 1.2 ARHGEF1 (Rho Guanine Nucleotide Exchange Factor 1) 8 1.2.1 Function and mechanism of ARHGEF1................8 1.2.2 Disease relative to ARHGEF1......................9 1.3 Lipid metabolism and antiviral response............10 1.3.1 Virus-induced lipid metabolism...................10 1.3.2 Type one interferon and lipid metabolism.........11 1.4 Aims...............................................11 Chapter 2 Materials and Methods........................12 2.1 Cell lines.........................................12 2.2 CRISPR/Cas9 system knock out ARHGEF1...............12 2.3 Mice...............................................13 2.4 Bone marrow derived macrophages (BMDMs) isolation..13 2.5 Virus infection....................................14 2.6 Ligand stimulation.................................14 2.7 qRT-PCR............................................15 2.7 Western blotting...................................16 2.8 RNA sequencing.....................................17 2.9 Luciferase assay...................................17 2.10 Oil red O staining................................17 Chapter 3 Results......................................19 3.1 CRSPR/Cas9 knock out ARHGEF1 in Huh-7 cells........19 3.2 IFNB1 expression is significantly increased in ARHGEF1-/- Huh-7 cells with virus infection.............19 3.3 ARHGEF1 negatively regulates RLR-mediated type I interferon..............................................20 3.4 Reconstitution of ARHGEF1 into ARHGEF1-/- Huh-7 cells suppresses the expression of IFNB1......................21 3.5 Reconstitution of ARHGEF1 critical domain: RGS domain compared with ΔRGS domain into ARHGEF1-/- Huh-7 cells suppresses the expression of IFNB1......................22 3.6 ARHGEF1 overexpression negatively regulates the activation of MAVS-mediated IFNB1 luciferase reporter in HEK293T.................................................22 3.7 ARHGEF1 deficiency leads to the increase of genes involved in antiviral responses.........................23 3.8 Ifnb1 expression is significantly increased in ARHGEF1+/- bone marrow derived macrophage (BMDM) with liposome-encapsulated poly (I:C) stimulation............24 3.9 Ifnb1 expression is significantly increased in ARHGEF1+/- serum with poly (I:C) stimulation............25 3.10 ARHGEF1 deficiency reduces the number of lipid droplets and lipid metabolism in Huh-7..................25 Chapter 4 Summary......................................27 Chapter 5 Discussion...................................28 Chapter 6 Figures......................................32 Chapter 7 Table........................................56 Chapter 8 Appendix.....................................57 Reference...............................................60 | |
| dc.language.iso | en | |
| dc.title | 探討ARHGEF1調節RLR所引起的第一型干擾素表現的能力 | zh_TW |
| dc.title | Determining the regulatory role of ARHGEF1 in RIG-I-like receptor (RLR)-mediated type I interferon expression. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐立中(Li-Chung Hsu),劉旻禕(Helene Minyi Liu) | |
| dc.subject.keyword | 第一型干擾素,ARHGEF1負調控,RLR調控路徑,防禦病毒的免疫反應,油滴, | zh_TW |
| dc.subject.keyword | Type I interferon,ARHGEF1,RLR signaling pathway,Anti-viral response,Lipoid droplets, | en |
| dc.relation.page | 66 | |
| dc.identifier.doi | 10.6342/NTU201801659 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2018-07-23 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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