【摘要】：氣道由氣管和各級支氣管組成,是人體對抗外界刺激的第一道防線。機械劃傷作為體外氣道受損模型的主要刺激因素,致使氣道上皮細胞出現(xiàn)極化改變、細胞壞死、分泌炎性細胞因子等變化。這個過程在體內(nèi)不斷反復,可遷延為氣道的慢性炎癥,是各種肺部疾病(COPD、慢性肺纖維化、肺癌等)的發(fā)生基礎。大量研究表明,細胞中NF-κB(nuclearfactor-κB)信號活化參與細胞的生長與增殖、凋亡以及炎癥反應中相關基因的轉(zhuǎn)錄,是炎癥反應發(fā)生的核心環(huán)節(jié),與機械劃傷引起的氣道炎癥有著密切關聯(lián)。然而,關于機械劃傷引起氣道炎性反應的確切機制,至今研究甚少。 NF-κB與抑制蛋白IκB關系緊密,IκB是NF-κB活化的重要開關。通常,靜息細胞中,IκB與NF-κB二聚體結(jié)合成NF-κB/IκB復合物,該復合物定位于細胞胞質(zhì),使NF-κB處于無活性狀態(tài)。當外界損傷刺激作用于細胞,IκB被激活,與NF-κB二聚體解離,NF-κB活性因此被迅速啟動,其p65亞基轉(zhuǎn)位入細胞核并進一步與其靶基因結(jié)合,增強炎癥相關基因的轉(zhuǎn)錄。p120-catenin(以下簡稱p120)是在篩選src癌蛋白底物時發(fā)現(xiàn)的,屬于連環(huán)蛋白家族新成員,以往的研究集中于p120介導細胞粘附及腫瘤發(fā)生。最近有報道,p120與皮膚炎癥、涎腺組織的炎癥及炎癥性腸病等相關,相關機制可能是通過NF-κB信號通路實現(xiàn),但p120與氣道炎癥的關系及其確切機制,國內(nèi)外研究甚少。因此,我們的研究圍繞機械劃傷致氣道炎癥中p120的活性變化展開,并進一步探討氣道炎癥中,p120與NF-κB信號通路的關系。 目的 本實驗采用機械劃傷培養(yǎng)的單層氣道上皮細胞建立體外氣道上皮損傷的模型,初步探討在損傷前后p120的蛋白表達量變化及對于NF-κB信號轉(zhuǎn)導途徑的影響,為深入探討氣道炎癥的發(fā)生發(fā)展機制提供理論與實驗依據(jù)。 方法 使用機械劃傷培養(yǎng)的單層氣道上皮細胞建立體外氣道上皮損傷的模型,利用Westernblot方法分別檢測機械劃傷前后p120、NF-κB亞基p65、抑制蛋白IκBα蛋白表達的變化,然后運用細胞核、漿分離提取蛋白技術(shù)觀察NF-κB亞基p65的核轉(zhuǎn)位情況,最后,在細胞上清液中,應用ELISA實驗(定量酶聯(lián)免疫吸附實驗)檢測機械劃傷前后NF-κB靶基因之一——炎癥細胞因子IL-8表達水平的變化。 結(jié)果 采用Westernblot檢測技術(shù),我們發(fā)現(xiàn)p120在人氣道上皮細胞的表達以分子量分別為120KD和100KD的亞型1、3為主,并且p120亞型3的蛋白表達量明顯高于亞型1;與未給予機械劃傷的正常對照組相比,機械劃傷后p120蛋白的表達逐漸減少,相反,NF-κB亞基p65被活化,其蛋白表達量在機械劃傷后增多,同時檢測到NF-κB的重要開關——IκBα蛋白表達在機械劃傷后減少。推測NF-κB的亞基p65的活化是通過IκBα的磷酸化并進一步降解而實現(xiàn)的,p120表達減少可激活NF-κB信號通路。核漿分離提取技術(shù)發(fā)現(xiàn)在機械劃傷后,細胞核中出現(xiàn)NF-κB亞基p65蛋白的表達,并且其細胞核表達量隨著機械劃傷后時間的延長而增多。最后運用ELISA檢測機械劃傷前后NF-κB靶基因之一——炎癥細胞因子IL-8的表達,發(fā)現(xiàn)機械劃傷后IL-8蛋白的分泌量較正常對照組增多,統(tǒng)計學結(jié)果顯示差異具有顯著性(p㩳0.05)。 結(jié)論 1.機械劃傷可抑制氣道上皮細胞中p120的表達,NF-κB信號通路活化。 2.機械劃傷后,NF-κB信號通路活化,可能是通過其活性“開關”——抑制蛋白IκBα的降解而實現(xiàn)的。 3.機械劃傷后,NF-κB信號通路活化,p65亞基的核轉(zhuǎn)位促進下游靶基因IL-8的表達,參與炎癥反應的發(fā)生。 總結(jié) 在機械劃傷導致的氣道炎癥中,p120很可能是通過對NF-κB信號通路的負性調(diào)節(jié),最終實現(xiàn)對氣道炎癥的抑制作用。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R363

【共引文獻】

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