NCOR启动子甲基化对巨噬细胞炎症的调控作用

发布时间：2019-05-06 17:05

【摘要】：研究背景急性肺损伤(ALI)和急性呼吸窘迫症(ARDS)严重威胁着人民的生命安全。严重的感染(如脓毒症)是引起ALI/ARDS主要原因之一。尽管目前对ALI/ARDS的研究取得了一定进展,但仍无有效的药物及治疗措施,其死亡率仍居高不下。因此深入探究ALI/ARDS炎症反应机制寻找新的ALI/ARDS的防治靶点显得尤为重要和迫切。国内外研究表明在急性肺损伤发生、发展过程中肺泡巨噬细胞发挥着重要作用。因此,深入研究巨噬细胞在这一过程中的作用有望为ALI/ARDS的防治提供新策略、手段。核受体协同抑制因子(nuclear receptor corepressor,NCOR)是一种核转录抑制因子。研究显示NCo R可通过TLR4信号转导或抑制NF-κB和AP-1调节炎症。但NCOR如何被调控,其机制尚不清楚。DNA甲基化是高等真核生物基因组中的主要表观遗传修饰,其在DNA甲基转移酶(DNMTs)催化下参与基因调控从而引起疾病的发生发展。本课题拟采用脂多糖(LPS)诱发的巨噬细胞炎症模型,探讨NCOR在炎症反应中的作用及其调控机制。研究目的:1.阐明NCOR在LPS诱导炎症反应中的作用;2.探讨NCOR启动子甲基化的机制以及在炎症反应中的作用。研究方法:1.LPS刺激巨噬细胞对NCOR的表达及炎症的影响用1ug/ml的LPS分别处理小鼠巨噬细胞RAW264.7 24h和48h,Western blot检测NCOR蛋白的表达水平,Real time-PCR检测NCOR、TNF-α、IL-6 mRNA水平。荧光素酶报告基因检测NF-κB启动子活性。2.LPS刺激巨噬细胞对NCOR基因启动子甲基化的影响LPS刺激细胞48h后,应用MSP检测NCOR启动子甲基化情况,Western blot检测DNMT3b的表达变化。Real time-PCR检测5’-aza和LPS联合处理细胞后NCOR mRNA的表达水平。3.下调DNMT3b对NCOR及炎症的影响巨噬细胞转染DNMT3b siRNA后,再用LPS处理巨噬细胞,分别应用Western blot和Real time-PCR检测DNMT3b的表达水平,以及DNMT3b siRNA和LPS联合作用下NCOR、TNF-α、IL-6的表达水平和NF-κB的启动子活性。结果:1.LPS刺激细胞24、48h后,NCOR蛋白和mRNA表达显著下调(p0.05);同时炎症因子TNF-α、IL-6 mRNA表达水平、NF-κB的启动子活性显著上升(p0.05)。2.LPS可诱导NCOR启动子发生甲基化,DNMT3b蛋白的表达水平升高,用5’-aza和LPS联合处理细胞后NCOR mRNA水平较LPS组有显著上升(p0.05)。3.DNMT3b siRNA可显著下调DNMT3b蛋白和mRNA水平,升高NCOR表达水平,从而抑制TNF-α、IL-6的表达水平和NF-κB的启动子活性(p0.05)。结论:1.LPS刺激巨噬细胞引起NCOR表达水平降低,促进炎症。2.LPS通过DNMT3b介导NCOR的启动子甲基化上调巨噬细胞炎症反应。3.下调DNMT3b表达可通过降低NCOR启动子甲基化水平进而抑制巨噬细胞炎症反应。
[Abstract]:Background Acute lung injury (ALI) and acute respiratory distress (ARDS) seriously threaten the lives of people. Severe infections, such as sepsis, are one of the main causes of ALI/ARDS. Although some progress has been made in the study of ALI/ARDS, there are still no effective drugs and treatments, and the mortality rate is still high. Therefore, it is very important and urgent to explore the mechanism of ALI/ARDS inflammation and find a new target for prevention and treatment of ALI/ARDS. Studies at home and abroad have shown that alveolar macrophages play an important role in the development of acute lung injury. Therefore, in-depth study of the role of macrophages in this process is expected to provide a new strategy and means for the prevention and treatment of ALI/ARDS. Nuclear receptor coinhibitor (nuclear receptor corepressor,NCOR is a nuclear inhibitor of transcription. Studies have shown that NCo R can regulate inflammation through TLR4 signal transduction or inhibition of NF- kappa B and AP-1. NCOR methylation is the main epigenetic modification in the genome of higher eukaryotes, and it is involved in gene regulation under the catalysis of DNA methyltransferase (DNMTs), which leads to the occurrence and development of diseases. In this study, we used lipopolysaccharide (LPS)-induced macrophage inflammation model to explore the role of NCOR in inflammatory response and its regulatory mechanism. Research purposes: 1. To clarify the role of NCOR in the inflammatory response induced by LPS; 2. To explore the mechanism of NCOR promoter methylation and its role in inflammatory reaction. Methods: the effects of 1.LPS stimulated macrophages on the expression of NCOR and inflammation were studied. RAW264.7 was treated with 1ug/ml LPS for 24 h and 48 h, respectively. Western blot was used to detect the expression of NCOR protein and Real time-PCR was used to detect NCOR,TNF- 伪. IL-6 mRNA level. NF- 魏 B promoter activity was detected by luciferase reporter gene assay. 2. Effects of LPS-stimulated macrophages on methylation of NCOR gene promoter in LPS-stimulated cells 48 hours later, NCOR promoter methylation was detected by MSP. The expression of DNMT3b was detected by Western blot. The expression level of NCOR mRNA was detected by Real time-PCR after co-treatment with 5'-aza and LPS. 3. Down-regulate the effect of DNMT3b on NCOR and inflammation after macrophages were transfected with DNMT3b siRNA, then treated with LPS, the expression of DNMT3b was detected by Western blot and Real time-PCR respectively, and NCOR,TNF- 伪 was induced by DNMT3b siRNA and LPS. IL-6 expression level and NF- 魏 B promoter activity. Results: after stimulated by 1.LPS for 24 h and 48 h, the expression of NCOR protein and mRNA were significantly down-regulated (p0.05). At the same time, the expression of TNF- 伪 and IL-6 mRNA and the activity of NF- 魏 B promoter increased significantly (p0.05). 2.LPS induced the methylation of NCOR promoter and the expression level of DNMT3b protein. After treated with 5'-aza and LPS, the level of NCOR mRNA was significantly higher than that of LPS group (p0.05). 3.DNMT3b siRNA significantly down-regulated the levels of DNMT3b protein and mRNA, increased the expression of NCOR, and thus inhibited the expression of TNF- 伪. IL-6 expression level and NF- 魏 B promoter activity (p0.05). Conclusion: lipopolysaccharide (1.LPS) stimulates macrophages to decrease the expression of NCOR and promote inflammation. 2.LPS mediated NCOR promoter methylation through DNMT3b to up-regulate macrophage inflammatory reaction. Down-regulation of DNMT3b expression can inhibit macrophage inflammatory response by decreasing methylation level of NCOR promoter.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

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