p120在机械划伤致气道上皮细胞炎症反应中对NF-κB信号通路的影响

发布时间：2018-09-11 18:17

【摘要】：气道由气管和各级支气管组成,是人体对抗外界刺激的第一道防线。机械划伤作为体外气道受损模型的主要刺激因素,致使气道上皮细胞出现极化改变、细胞坏死、分泌炎性细胞因子等变化。这个过程在体内不断反复,可迁延为气道的慢性炎症,是各种肺部疾病(COPD、慢性肺纤维化、肺癌等)的发生基础。大量研究表明,细胞中NF-κB(nuclearfactor-κB)信号活化参与细胞的生长与增殖、凋亡以及炎症反应中相关基因的转录,是炎症反应发生的核心环节,与机械划伤引起的气道炎症有着密切关联。然而,关于机械划伤引起气道炎性反应的确切机制,至今研究甚少。 NF-κB与抑制蛋白IκB关系紧密,IκB是NF-κB活化的重要开关。通常,静息细胞中,IκB与NF-κB二聚体结合成NF-κB/IκB复合物,该复合物定位于细胞胞质,使NF-κB处于无活性状态。当外界损伤刺激作用于细胞,IκB被激活,与NF-κB二聚体解离,NF-κB活性因此被迅速启动,其p65亚基转位入细胞核并进一步与其靶基因结合,增强炎症相关基因的转录。p120-catenin(以下简称p120)是在筛选src癌蛋白底物时发现的,属于连环蛋白家族新成员,以往的研究集中于p120介导细胞粘附及肿瘤发生。最近有报道,p120与皮肤炎症、涎腺组织的炎症及炎症性肠病等相关,相关机制可能是通过NF-κB信号通路实现,但p120与气道炎症的关系及其确切机制,国内外研究甚少。因此,我们的研究围绕机械划伤致气道炎症中p120的活性变化展开,并进一步探讨气道炎症中,p120与NF-κB信号通路的关系。目的本实验采用机械划伤培养的单层气道上皮细胞建立体外气道上皮损伤的模型,初步探讨在损伤前后p120的蛋白表达量变化及对于NF-κB信号转导途径的影响,为深入探讨气道炎症的发生发展机制提供理论与实验依据。方法使用机械划伤培养的单层气道上皮细胞建立体外气道上皮损伤的模型,利用Westernblot方法分别检测机械划伤前后p120、NF-κB亚基p65、抑制蛋白IκBα蛋白表达的变化,然后运用细胞核、浆分离提取蛋白技术观察NF-κB亚基p65的核转位情况,最后,在细胞上清液中,应用ELISA实验(定量酶联免疫吸附实验)检测机械划伤前后NF-κB靶基因之一——炎症细胞因子IL-8表达水平的变化。结果采用Westernblot检测技术,我们发现p120在人气道上皮细胞的表达以分子量分别为120KD和100KD的亚型1、3为主,并且p120亚型3的蛋白表达量明显高于亚型1;与未给予机械划伤的正常对照组相比,机械划伤后p120蛋白的表达逐渐减少,相反,NF-κB亚基p65被活化,其蛋白表达量在机械划伤后增多,同时检测到NF-κB的重要开关——IκBα蛋白表达在机械划伤后减少。推测NF-κB的亚基p65的活化是通过IκBα的磷酸化并进一步降解而实现的,p120表达减少可激活NF-κB信号通路。核浆分离提取技术发现在机械划伤后,细胞核中出现NF-κB亚基p65蛋白的表达,并且其细胞核表达量随着机械划伤后时间的延长而增多。最后运用ELISA检测机械划伤前后NF-κB靶基因之一——炎症细胞因子IL-8的表达,发现机械划伤后IL-8蛋白的分泌量较正常对照组增多,统计学结果显示差异具有显著性(p㩳0.05)。结论 1.机械划伤可抑制气道上皮细胞中p120的表达,NF-κB信号通路活化。 2.机械划伤后,NF-κB信号通路活化,可能是通过其活性“开关”——抑制蛋白IκBα的降解而实现的。 3.机械划伤后,NF-κB信号通路活化,p65亚基的核转位促进下游靶基因IL-8的表达,参与炎症反应的发生。总结在机械划伤导致的气道炎症中,p120很可能是通过对NF-κB信号通路的负性调节,最终实现对气道炎症的抑制作用。
[Abstract]:The airway is composed of trachea and bronchi, which is the first line of defense against external stimuli. Mechanical scratch, as the main stimulus factor in the model of airway damage in vitro, causes changes in airway epithelial cells such as polarization, cell necrosis, and secretion of inflammatory cytokines. Sexual inflammation is the basis of various pulmonary diseases (COPD, chronic pulmonary fibrosis, lung cancer, etc.). Numerous studies have shown that activation of nuclear factor-kappa B (NF-kappa B) signal in cells is involved in cell growth and proliferation, apoptosis and the transcription of related genes in inflammatory response, which is the core link of inflammation and airway inflammation caused by mechanical scratch. Symptoms are closely related. However, the exact mechanism by which mechanical scratches cause airway inflammation has been poorly studied.
NF-kappa B is closely related to inhibitor I-kappa B, and I-kappa B is an important switch for activation of NF-kappa B. Usually, in resting cells, I-kappa B binds to the dimer of NF-kappa B to form a complex of NF-kappa B/I-kappa B, which is located in the cytoplasm of cells and makes NF-kappa B inactive. P120-catenin (p120) is a new member of the catenin family found in screening SRC oncoprotein substrates. Previous studies have focused on p120-mediated cell adhesion and tumorigenesis. It has been reported that p120 is associated with skin inflammation, salivary gland inflammation and inflammatory bowel disease. The related mechanism may be realized by NF-kappa B signaling pathway, but the relationship between p120 and airway inflammation and its exact mechanism has not been studied at home and abroad. Objective to investigate the relationship between p120 and NF- kappa B signaling pathway in airway inflammation.
objective
In this study, a single-layer airway epithelial cells cultured by mechanical scratch were used to establish an in vitro model of airway epithelial injury. The changes of p120 protein expression before and after injury and its effect on NF-kappa B signal transduction pathway were preliminarily investigated, which provided theoretical and experimental basis for further study of the mechanism of airway inflammation.
Method
The expression of p120, NF-kappa B subunit p65 and inhibitor of protein I-kappa B alpha were detected by Western blot. The nuclear translocation of NF-kappa B subunit p65 was observed by nuclear and plasma isolation techniques. Then, the expression of inflammatory cytokine IL-8, one of the target genes of NF-kappa B, was detected by ELISA (quantitative enzyme-linked immunosorbent assay) before and after mechanical scratch.
Result
Using Western blot, we found that the expression of p120 in human airway epithelial cells was dominated by subtype 1,3 with molecular weight of 120 KD and 100 KD, respectively, and the expression of p120 subtype 3 was significantly higher than that of subtype 1. The expression of p65 increased after mechanical scratch, and the expression of I-kappa B alpha, an important switch of NF-kappa B, decreased after mechanical scratch. It was speculated that the activation of p65, a subunit of NF-kappa B, was achieved by the phosphorylation and degradation of I-kappa B alpha. The decrease of p120 expression could activate the NF-kappa B signaling pathway. It was found that the expression of NF-kappa B subunit p65 protein in the nucleus increased with the prolongation of the time after mechanical scratch. Finally, the expression of inflammatory cytokine IL-8, one of the target genes of NF-kappa B, was detected by ELISA, and the secretion of IL-8 protein was higher after mechanical scratch than that after mechanical scratch. Normal control group increased, statistical results showed significant difference (P? 0.05).
conclusion
1. mechanical scratches can inhibit the expression of p120 in airway epithelial cells and activate the NF- kappa B signaling pathway.
2. After mechanical scratch, the activation of NF-kappa B signaling pathway may be achieved by its active "switch" - inhibiting the degradation of protein I-kappa B alpha.
3. After mechanical scratch, NF-kappa B signaling pathway is activated, and nuclear translocation of p65 subunit promotes the expression of downstream target gene IL-8 and participates in the occurrence of inflammation.
summary
In the airway inflammation caused by mechanical scratch, p120 is likely to inhibit airway inflammation through negative regulation of NF-kappa B signaling pathway.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【共引文献】