Elafin真核表达载体的构建及其对气道粘液高分泌的调节

目的:构建天然内生多肽Elafin真核表达载体,探讨其对气道粘液高分泌的影响。方法:抽提Elafin行RT-PCR获取Elafin cDNA,双酶切后将片段装载到pMD18-T载体上。以pMD18-T-Elafin为模板行PCR反应,产物胶回收并双酶切后定向克隆至pEGFP-N1上,转化,筛选,双酶切鉴定重组质粒。将pEGFP-N1-Elafin转染正常人支气管上皮细胞HBE16,给予脂多糖(LPS)刺激,Western blot检测细胞内Elafin蛋白的相对含量,RT-PCR检测各组Elafin mRNA和粘蛋白(MUC)5AC mRNA表达水平,荧光素酶报告基因检测系统测定核转录因子-κB(NFκ-B)的活性;ELISA法分析各组细胞MUC5AC蛋白的相对含量。结果:成功构建Elafin真核表达载体,转染重组Elafin的HBE16细胞成功表达Elafin蛋白。LPS刺激可增强NF-κB的活性,该活性在转染重组Elafin后显著降低;MUC5AC蛋白含量及mRNA水平在LPS刺激后也显著升高,在转染重组Elafin后二者的表达水平明显降低。结论:Elafin真核表达载体成功构建,初步发现Elafin可通过降低NFκ-B的活性来下调MUC5AC的表达,为进一步深入研究其对气道粘液高分泌的调节机制奠定了基础。     

BRAP对LPS诱导的人气道上皮细胞系黏液高分泌的影响

目的探讨蛙皮素受体激活蛋白(BRAP)对脂多糖(LPS)诱导的气道黏液高分泌的影响及相关作用机制。方法体外培养人气道上皮HBE16细胞,转染已构建好的p EGFP-N1-BRAP,以空质粒载体作为对照,给予LPS刺激,同时分别以ERK抑制剂U0126、JNK抑制剂SP600125、P38抑制剂SB203580干预。观察细胞内活性氧(ROS)含量、黏蛋白(MUC)5AC表达和核因子-κB(NF-κB)活性以及细胞活力的变化。结果转染p EGFP-N1-BRAP后ROS明显减少(P0.01);同单纯LPS刺激相比,MUC5AC蛋白含量和mRNA水平在转染p EGFP-N1-BRAP后明显降低(P0.01),NF-κB活性亦呈相同趋势(P0.05);在U0126组,MUC5AC的表达较转染重组质粒组显著降低(P0.01),NF-κB活性也显著下调(P0.05)。结论 BRAP可以通过ROS/ERK/MAPK信号通路阻止胞质内NF-κB的活化,从而下调MUC5AC的生成。     

转染重组人Elafin基因保护NCI-H292细胞系

目的探讨转染重组人白细胞弹性蛋白酶抑制剂内生多肽(Elafin)基因保护气道上皮的分子机制。方法构建人Elafin重组质粒pEGFP-C1-Elafin,并转染入人气道黏膜上皮细胞系NCI-H292细胞,将其与多形核粒细胞(PMN)共孵育,经脂多糖(LPS)刺激24 h后,用Western blot检测细胞中紧密连接相关蛋白ZO-1的表达及MTT法测定细胞与胶原的附着能力。结果经LPS刺激后,Western blot及MTT法发现,与对照组和转染重组质粒组相比,转染空载体的NCI-H292细胞中ZO-1的表达及细胞与胶原附着能力均明显降低(P<0.01);而与对照组相比,转染重组质粒的细胞中ZO-1的表达及细胞与胶原附着能力均未见明显下降。结论通过转染重组人Elafin可保护气道上皮,增强气道抵抗炎性损伤能力。     

小窝蛋白1 对气道上皮细胞黏液高分泌的影响

目的:探讨小窝蛋白1(Cav-1)对脂多糖(LPS)诱导的气道黏液高分泌的影响。方法:体外培养人气道上皮细胞(16HBE),用LPS刺激细胞构建黏液高分泌模型,以Toll样受体4(TLR4)抑制剂E5564、核转录因子-κB(NF-κB)抑制剂PDTC、转染Cav-1质粒和siRNA为干预因素,将细胞随机分为对照组、LPS刺激组、LPS+Cav-1质粒组、LPS+Cav-1 siRNA组、LPS+阴性siRNA组、LPS+空质粒组、LPS+E5564组及LPS+PDTC组。四甲基偶氮唑盐法(MTT)检测各组细胞的活力;RT-PCR检测黏蛋白(MUC)5AC的转录水平;Western blot检测Cav-1、TLR4、磷酸化IκBα(p-IκBα)蛋白的相对含量;ELISA检测MUC5AC的分泌水平;激光共聚焦技术检测细胞内MUC5AC蛋白的分布和含量。结果:LPS刺激组细胞内TLR4、p-IκBα、NF-κB、MUC5AC转录及蛋白水平显著高于对照组(P值均0.05),过表达Cav-1可进一步增加上述指标的表达量,而下调Cav-1及给予E5564、PDTC可以抑制LPS引起的上述效应(P0.05)。结论:Cav-1可通过上调TLR4/NF-κB信号通路而加重LPS诱导的MUC5AC的表达量。     

HSP72过度表达对脂多糖刺激的巨噬细胞中NF-κB活性的影响

目的研究过度表达的热休克蛋白72(heat shock protein,HSP72)对脂多糖(lipopolysaccha-ride,LPS)刺激的巨噬细胞中NF-κB活性的影响.方法构建pCD-hsp72重组质粒,转染小鼠巨噬细胞RAW264.7,G418筛选稳定转染hsp72基因的细胞,Western印迹检测转染细胞HSP72的表达水平及LPS刺激后RAW264.7细胞内HSP72的表达水平、LPS刺激后转染细胞中IκBα的含量和胞核中NF-κB的含量的变化.结果构建pCD-hsp72重组质粒并转入巨噬细胞,获得稳定转染hsp72基因的细胞株,LPS刺激细胞内HSP72表达增加;HSP72可通过减少LPS刺激的巨噬细胞中IκBα的降解而抑制NF-κB活性.结论HSP72能抑制LPS激活的巨噬细胞中NF-κB的活化.     

槲皮素基于EGFR信号通路减轻大鼠气道黏液高分泌北大核心CSCD

目的:探讨槲皮素对LPS诱导的大鼠气道黏液高分泌的作用及其分子机制。方法:将36只SD大鼠随机分为:对照组、LPS组、槲皮素低、中、高剂量组、AG1478组;气道内滴注LPS建立大鼠气道黏液高分泌模型。HE染色观察肺组织的病理变化;AB-PAS染色观察杯状细胞增生和黏液分泌情况;ELISA检测肺组织中MUC5AC浓度;采用免疫组织化学法(IHC)检测MUC5AC、EGFR、PKC、NF-κB表达;RT-qPCR检测肺组织MUC5AC及EGFR mRNA表达;Western blot检测p-EGFR、EGFR、p-PI3K、PI3K、p-PKC、PKC、p-AKT、AKT、NF-κB表达。结果:与LPS组相比,槲皮素低、中、高剂量组病理变化逐渐减轻,肺组织炎症积分和AB-PAS阳性着色面积逐渐降低(P<0.05);槲皮素能减少肺组织MUC5AC、EGFR、PKC、NF-κB表达,降低MUC5AC含量,并下调MUC5AC及EGFR的mRNA水平(P<0.05);槲皮素能减少p-EGFR/EGFR、p-PI3K/PI3K、p-PKC/PKC、p-AKT/AKT、NF-κB蛋白表达(P<0.05)。结论:气管内滴注LPS能成功诱导大鼠气道黏液高分泌模型,槲皮素对LPS诱导大鼠气道黏液高分泌具有拮抗作用,其作用机制可能与抑制EGFR信号通路,调控p-EGFR/EGFR、p-PI3K/PI3K、p-PKC/PKC、p-AKT/AKT、NF-κB表达,下调MUC5AC表达有关。     

TNF-α与香烟烟雾在诱导气道上皮细胞株黏蛋白表达的相互作用

目的 探讨肿瘤坏死因子TNF-α与香烟提取物共同诱导气道黏液高分泌的相互关系及作用特点。方法 培养的人气道上皮细胞BEAS-2B,转染核转录因子（NF）-κB"decoy"寡核苷酸（ODNs）,并以乱序NF-κB ODNs为对照转染组,各组均分别予以TNF-α、10%香烟提取物（CSE）单独刺激及共同刺激,以Western blot、ELISA及RT-PCR法检测各组刺激前后磷酸化表皮生长因子受体（p-EGFR）、黏蛋白（MUC5AC）蛋白及mRNA水平。结果 转染乱序NF-κB ODNs的细胞予以TNF-α、10% CSE刺激后,各组细胞中p-EGFR蛋白水平较对照组升高,伴随MUC5AC蛋白含量及基因转录水平的提高(P<0.05);共孵育组中较单独刺激组升高更为显著（P<0.05）。转染NF-κB"decoy"ODNs组再予以TNF-α刺激,细胞中MUC5AC蛋白含量及mRNA水平与未转染组相比升高不明显,而单用CSE刺激组中的MUC5AC、p-EGFR水平仍有明显升高（P<0.05）;共孵育组显示出与CSE单独刺激组相似的结果。结论 TNF-α、CSE能协同促进气道上皮细胞中黏蛋白合成,转录因子NF-κB主要参与TNF-α所致的MUC5AC表达,而在CSE诱导的效应过程中作用不明显。     

脂多糖诱导人气道上皮细胞hBD-2表达及核转录因子κB活性的变化

 目的：观察脂多糖（LPS）诱导人气道上皮细胞人β防御素-2（hBD-2）的表达及核转录因子κB(NF-κB)活性的变化，探讨NF-κB在LPS诱导人气道上皮细胞hBD-2表达中的作用。方法: 体外培养人气道原代上皮细胞，用不同剂量LPS刺激，RT-PCR法检测hBD-2 mRNA的表达，凝胶迁移试验（EMSA）检测不同时相NF-κB活性的变化。 结果: LPS刺激2 h后可见人气道上皮细胞hBD-2 mRNA表达，并呈时间、剂量依赖性；NF-κB在LPS刺激1h后明显活化，并与LPS剂量正相关，抗体超迁移率实验结果显示NF-κB的异型二聚体p65-p50参与了NF-κB的活化。 结论:一定剂量的LPS可诱导人气道上皮细胞hBD-2 mRNA表达，NF-κB在LPS诱导气道上皮细胞hBD-2 mRNA起重要的调控作用。     

肝X受体激动剂对脂多糖诱导的炎症反应相关因子IRAK-4和NF-κB的影响

目的:观察在枯否细胞中人工合成的肝X受体激动剂在脂多糖(LPS)诱导的炎症反应中,对白细胞介素-1受体相关激酶-4(IRAK-4)和核因子NF-κB的影响.方法:雄性昆明小鼠,用胶原酶原位灌注法分离和培养肝脏Kupffer细胞,获得的细胞随机分为正常对照组、 LPS处理组、 LXR人工合成激动剂T0901317处理组和LPS+T0901317共同处理组.实时-聚合酶链式反应和蛋白Western blot检测各组细胞LXR、 IRAK-4和NF-κB的 mRNA和蛋白表达水平;凝胶电泳迁移率(EMSA)检测NF-κB活性水平.结果:LXR mRNA和蛋白在T0901317组表达都是最高的,而在LPS处理组最低.IRAK4和NF-κB的mRNA和蛋白水平在LPS处理组最高,联合处理组要低于LPS处理组.NF-κB的活性在LPS最高,联合处理组和T0901317处理组都较LPS组降低.结论:LXR激动剂能有效的上调LXR基因和蛋白水平,同时抑制TLR4信号通路中IRAK-4和NF-κB的mRNA水平和蛋白表达水平,并且能抑制NF-κB的活化.     

Ezrin介导中性粒细胞弹性蛋白酶诱导的人气道上皮细胞株分泌黏蛋白增加

目的探讨膜连接蛋白Ezrin在中性粒细胞弹性蛋白酶(NE)诱导气道黏液高分泌的作用及相关调节机制。方法以中性粒细胞弹性蛋白酶刺激培养的人气道上皮HBE16细胞,ELISA法、real-time PCR法测定黏蛋白(MUC)5AC的蛋白及mRNA水平。免疫荧光法检测Ezrin蛋白含量。结果 NE刺激30 min后MUC5AC mRNA表达增强,细胞中及培养上清中黏蛋白MUC5AC的含量均显著高于对照组(P0.01);转染Ezrin永久磷酸化载体pEGFPN1-Ezrin-T567D的细胞经NE刺激后,胞质内磷酸化Ezrin表达明显增强,且胞膜分布增多。同时,细胞培养上清液中的MUC5AC蛋白含量亦显著高于单纯NE组(P0.01)。遏制Ezrin磷酸化pEGFP-N1-Ezrin-T567A载体转染组在NE刺激后,Ezrin蛋白与pEGFP-N1-Ezrin-T567D组相比有明显减少,且未出现向胞膜聚集的趋势,同时分泌至培养上清的MUC5AC蛋白也有明显减少(P0.01),同时伴随胞质内MUC5AC蛋白有所增加(P0.05)。结论 Ezrin蛋白参与了NE诱导的MUC5AC蛋白分泌,是气道上皮细胞MUC5AC分泌的重要调控分子。     

Prior airway exposure to allergen increases virus-induced airway hyperresponsiveness

BACKGROUND: Respiratory syncytial virus (RSV) bronchiolitis in early life can lead to changes in airway function, but there are likely additional predisposing factors, such as prior allergen exposure, determining which children develop wheezing and asthma. OBJECTIVE: To define the effects of prior airway exposure to sensitizing allergen on the development of airway inflammation and hyperresponsiveness (AHR) to subsequent RSV infection. METHODS: BALB/c mice were exposed to ovalbumin or PBS exclusively through the airways and subsequently infected with RSV or sham-inoculated. AHR, lung inflammation, and the frequency of cytokine-producing T lymphocytes in the lung were determined. RESULTS: In PBS-exposed mice, RSV infection induced AHR and an increased proportion of TH1-type (IFN-gamma and IL-12) cytokine-producing cells in the lungs. However, in mice previously exposed to ovalbumin through the airways and subsequently infected with RSV, the degree of AHR was significantly increased and was associated with an increased proportion of TH2 (IL-4, IL-5) cytokine-producing T lymphocytes. This response was also associated with an increased accumulation of eosinophils, neutrophils, and CD8+ T cells in the lungs. CONCLUSIONS: These data suggest that prior airway exposure to allergen may predispose sensitized hosts to a greater degree of altered airway function upon subsequent respiratory viral infection.     

Ultrastructural changes in rat airway epithelium in asthmatic airway remodeling

ObjectivesTo explore asthmatic rat airway epithelial cells mitochondrial ultrastructure changes.MethodsSix female Wistar rats of the same weight (60–80 g) were randomly divided into two groups: the asthmatic group and the control group. According to the OVA inhaled method, the asthmatic airway remodeling rat model was established. Epithelial tissue of the rat trachea was taken from the two groups for transmission electron microscopy (TEM); we counted the number of mitochondria and observed the airway ciliated epithelium, intercellular collagen deposition in the two rat groups and mitochondrial ultrastructure change.ResultsAirway multilayer ciliated epithelium develops, with cilia fallen off; goblet cells increased and irregular, mitochondrial basement membrane density is decreased, mitochondrial crista is reduced, and the nucleus has more incisures and irregular shape in asthmatic rats; airway epithelial cell matrix collagen deposition increased; and lamellar body and mitochondrial cavity formation.ConclusionsIn the asthmatic rat airway, epithelial cells undergo apoptosis and the numbers of mitochondria increased compared with the ones in normal rat airway but lose normal structure.     

Regulation of airway surface liquid volume by human airway epithelia

Mucus clearance on airway surfaces is a primary form of pulmonary defense. The efficiency of mucus clearance in large part depends on the volume of the airway surface liquid components, including both the periciliary liquid (PCL) layer and the mucus layer. Studies with in vitro model systems suggest that the mucus layer acts as a passive reservoir to redistribute water to and from, as needed, the PCL layer. In contrast, the overall volume of airway surface liquid is determined by active transepithelial salt transport. Data from in vitro systems suggest that airway epithelia have the capacity to both absorb and secrete liquid in response to the volume requirements on the apical surface. At present, the nature of the signals that transmit information about airway surface liquid volume to epithelia and their sensors are unknown. However, progress in elucidation of this system is important, because it appears that these systems are deranged in the genetic disease cystic fibrosis, which is characterized by airway surface liquid volume depletion, mucus stasis, and chronic infection. Thus, insights into these systems may offer novel therapeutic opportunities to correct this physiologic dysfunction of airway epithelia.     

Glutathione redox regulates airway hyperresponsiveness and airway inflammation in mice

Glutathione is the major intracellular redox buffer. We have shown that glutathione redox status, which is the balance between intracellular reduced (GSH) and oxidized (GSSG) glutathione, in antigen-presenting cells (APC) regulates the helper T cell type 1 (Th1)/Th2 balance due to the production of IL-12. Bronchial asthma is a typical Th2 disease. Th2 cells and Th2 cytokines are characteristic of asthma and trigger off an inflammation. Accordingly, we studied the effects of the intracellular glutathione redox status on airway hyperresponsiveness (AHR) and allergen-induced airway inflammation in a mouse model of asthma. We used gamma-Glutamylcysteinylethyl ester (gamma-GCE), which is a membrane-permeating GSH precursor, to elevate the intracellular GSH level and GSH/GSSG ratio of mice. In vitro, gamma-GCE pretreatment of human monocytic THP-1 cells elevated the GSH/GSSG ratio and enhanced IL-12(p70) production induced by LPS. In the mouse asthma model, intraperitoneal injection of gamma-GCE elevated the GSH/GSSG ratio of lung tissue and reduced AHR. gamma-GCE reduced levels of IL-4, IL-5, IL-10, and the chemokines eotaxin and RANTES (regulated on activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid, whereas it enhanced the production of IL-12 and IFN-gamma. Histologically, gamma-GCE suppressed eosinophils infiltration. Interestingly, we also found that gamma-GCE directly inhibited chemokine-induced eosinophil chemotaxis without affecting eotaxin receptor chemokine receptor 3 (CCR3) expressions. Taken together, these findings suggest that changing glutathione redox balance, increase in GSH level, and the GSH/GSSG ratio by gamma-GCE, ameliorate bronchial asthma by altering the Th1/Th2 imbalance through IL-12 production from APC and suppressing chemokine production and eosinophil migration itself.     

Irritant-induced airway disorders

Thousands of persons experience accidental high-level irritant exposures each year but most recover and few die. Irritants function differently than allergens because their actions proceed nonspecifically and by nonimmunologic mechanisms. For some individuals, the consequence of a single massive exposure to an irritant, gas, vapor or fume is persistent airway hyperresponsiveness and the clinical picture of asthma, referred to as reactive airways dysfunction syndrome (RADS). Repeated irritant exposures may lead to chronic cough and continual airway hyperresponsiveness. Cases of asthma attributed to repeated irritant-exposures may be the result of genetic and/or host factors.