Resistin对炎症信号途径的调控研究

发布时间：2018-02-15 07:04

本文关键词： 抗素环氧合酶-2 核因子-κB 炎症信号途径 C15肽　出处：《华中农业大学》2010年博士论文　论文类型：学位论文

【摘要】： Resistin称抗素(也称FIZZ3),是一种含有114个氨基酸的细胞因子。近来发现resistin具有一系列前炎症因子的特征,在炎症中能够诱导血管内皮细胞粘附分子的表达,从而导致白细胞向各个炎症组织中的浸润。尽管resistin在炎症发生中已有不少报道,但其与经典炎症信号途径的关系,在炎症中的作用及其调控机制尚未阐明。环氧合酶2(COX-2)是前列腺素合成中的一个限速酶。在人体中存在两种不同的COX异构体,分别称为COX-1和COX-2。其中COX-2是一种经过诱导而产生的酶,在正常生理条件下维持较低的表达水平,但在炎症部位表达上调,催化炎性PGs的合成并诱发炎症反应。此外,COX-2的启动子上存在NF-κB的结合元件,可受转录因子NF-κB调控。一些重要的炎症因子例如TNFa和IL-1可通过激活NF-κB而上调COX-2的表达。本研究以resistin为研究对象,采用real-time PCR、Western Blotting和细胞转染等技术,系统研究了resistin通过经典的炎症信号途径上调COX-2的基因表达及抗炎肽C15对resistin等引起的炎症反应的抑制作用分子机制,结果如下： 1.在巨噬细胞内分别转染猪和小鼠resistin的超表达载体,采用real-timePCR和Western Blotting方法检测细胞内源的COX-2的mRNA和蛋白质水平的表达变化。结果表明,resistin在RAW264.7巨噬细胞系中可以显著地上调COX-2的mRNA和蛋白质的表达。更重要的是,通过原核表达制备了抗resistin的抗体,用抗体处理可以抵消resistin的这种作用。 2.以巨噬细胞为模型,通过负显性IκBα突变体和PDTC这两种抑制物质研究了resistin对COX-2的信号转导途径的调控。结果表明,无论是超表达负显性IκBα突变体载体,还是用抑制剂PDTC刺激,都可以抑制resistin对COX-2的诱导作用。进一步的研究证实,resistin的超表达可以上调NF-κB分子中的p65亚基的表达,进而诱导COX-2的表达水平的上升。 3.以resistin对小鼠巨噬细胞作不同时间梯度的处理,用Western Blotting方法分析resistin对一种MAPKKK激酶TAK1 (Transforming growth factorβactivated kinase 1)的磷酸化程度的影响。结果表明,resistin可以显著上调TAK1的磷酸化水平。 4.以促炎物质resistin,LPS,PA分别和C15共同处理小鼠巨噬细胞,检测炎症基因COX-2和iNOS (inducible nitric oxide synthase)的表达变化。结果表明,炎症基因COX-2和iNOS可以被三种促炎物质诱导表达,并且这种诱导的情况可以显著被C15所抑制住。 5.在巨噬细胞中,以LPS在不同时间梯度刺激细胞,用细胞免疫荧光的方法检测NF-κB的p65亚基的入核情况。结果证实,C15可以明显抑制LPS所导致的p65的入核情况,表明C15是通过阻止NF-κB的p65亚基入核,从而抑制相关炎症因子的表达,进而表现出一定的抗炎活性。
[Abstract]:Resistin, also known as FIZZ3, is a 114 amino acid cytokine. Recently, resistin has been found to have a series of proinflammatory cytokines that induce the expression of adhesion molecules in vascular endothelial cells. Although resistin has been reported to be involved in inflammation, its relationship with the classical inflammatory signaling pathway, its role in inflammation and its regulatory mechanism have not been elucidated. Cyclooxygenase 2 (cyclooxygenase 2) is a rate-limiting enzyme in the synthesis of prostaglandins. There are two different COX isomers in the human body, called COX-1 and COX-2. COX-2 is an induced enzyme. The low expression level was maintained under normal physiological conditions, but up-regulated in inflammatory site, which catalyzed the synthesis of inflammatory PGs and induced inflammatory response. In addition, there were NF- 魏 B binding elements on the promoter of COX-2. Some important inflammatory factors such as TNFa and IL-1 can up-regulate the expression of COX-2 by activating NF- 魏 B. In this study, resistin was used to investigate the mechanism of resistin up-regulation of COX-2 gene expression through classical inflammatory signaling pathway and the inhibitory effect of anti-inflammatory peptide C15 on the inflammatory response induced by resistin by using real-time PCR Western Blotting and cell transfection techniques. The results are as follows:. 1. The superexpression vectors of porcine and mouse resistin were transfected into macrophages, respectively. The expression of mRNA and protein in endogenous COX-2 was detected by real-timePCR and Western Blotting. The results showed that the expression of mRNA and protein of COX-2 was significantly up-regulated in RAW264.7 macrophages. The antibody against resistin was prepared by prokaryotic expression, and the effect of resistin could be counteracted by antibody treatment. 2. Macrophage model was used to study the regulation of resistin signal transduction pathway in COX-2 by negative dominant I 魏 B 伪 mutants and PDTC. The results showed that both overexpression negative dominant I 魏 B 伪 mutant vector and inhibitor PDTC were used to stimulate the signal transduction pathway of COX-2. Further studies showed that the overexpression of resistin could up-regulate the expression of p65 subunit in NF- 魏 B and induce the increase of COX-2 expression. 3. The effect of resistin on the phosphorylation of a MAPKKK kinase TAK1 transforming growth factor 尾 activated kinase 1 was analyzed by Western Blotting. The results showed that resistin could significantly up-regulate the phosphorylation level of TAK1. 4. The expression of inflammatory genes COX-2 and iNOS inducible nitric oxide synthases were detected by co-treatment of mouse macrophages with proinflammatory substance resistinin-LPS-PA and C15, respectively. The results showed that the inflammatory genes COX-2 and iNOS could be induced by three kinds of proinflammatory substances. And this inducement can be significantly suppressed by C 15. 5. In macrophages, the entry of p65 subunit of NF- 魏 B was detected by cellular immunofluorescence with LPS at different time gradient. The results showed that C15 could significantly inhibit p65 entry induced by LPS. It is suggested that C15 inhibits the expression of related inflammatory factors by blocking the p65 subunit of NF- 魏 B into the nucleus and thus exhibits anti-inflammatory activity.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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