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Title: | 探討ARHGEF1調節RLR所引起的第一型干擾素表現的能力 Determining the regulatory role of ARHGEF1 in RIG-I-like receptor (RLR)-mediated type I interferon expression. |
Authors: | Ci-Yun Huang 黃頎勻 |
Advisor: | 江皓森(Hao-Sen Chiang) |
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, Type I interferon,ARHGEF1,RLR signaling pathway,Anti-viral response,Lipoid droplets, |
Publication Year : | 2018 |
Degree: | 碩士 |
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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19971 |
DOI: | 10.6342/NTU201801659 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生命科學系 |
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