地塞米松对人鼻黏膜上皮细胞前炎性因子表达的抑制作用

目的观察地塞米松对脂多糖(lipopolysac-charide,LPS)诱导的人鼻黏膜上皮细胞缺氧诱导因子-1(hypoxic inducible factor-1,HIF-1)及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达抑制情况及意义。方法无血清原代培养人鼻息肉和下鼻甲黏膜上皮细胞,以LPS100ng/ml,白细胞介素-1β(interleukin-1β,IL-1β)20ng/ml,LPS100ng/ml加地塞米松13ng/ml,IL-1β20ng/ml加地塞米松13ng/ml作用于对数生长期的上皮细胞3、6、9小时后,采用免疫细胞化学和原位杂交方法检测HIF-1α和VEGF蛋白及mRNA的表达情况。结果①LPS和IL-1β作用于上皮细胞时,HIF-1α及VEGF表达增加具有时间依赖性,以LPS100ng/ml6小时组最明显(P<0.05),且鼻息肉中的表达明显高于下鼻甲(P<0.05);②LPS100ng/ml加地塞米松13ng/ml组和IL-1β20ng/ml加地塞米松13ng/ml组表达强度分别较LPS100ng/ml组和IL-1β20ng/ml组弱(P<0.05)。两抑制组间表达无显著性差异。结论炎性因子及感染性因子可以诱导人鼻黏膜上皮细胞大量表达HIF-1α及VEGF,而地塞米松对其表达有很强的抑制作用。     

淋巴细胞过度活化及细胞因子与鼻息肉的免疫发病机制研究

目的：探讨淋巴细胞CD4、CD69和细胞因子RANTES、IL-5、IL-8在鼻息肉组织中的表达及其在鼻息肉发生、发展中的作用。方法：苏木精-伊红染色、免疫组织化学和图像分析法检测34例鼻息肉组织（鼻息肉组）和30例鼻甲黏膜组织（对照组）中CD4、CD69、CD34、RANTEs、IL-5、IL-8的表达。结果：鼻息肉组中淋巴细胞、嗜酸粒细胞弥漫浸润,腺体增生、杯状细胞增生、纤维增生、间质水肿的阳性率明显高于对照组（均P〈0．01）。鼻息肉组中CD4、CD69、IL-5、IL-8、RANTES阳性淋巴细胞数密度（n／mm^2）明显高于对照组（均P〈0．05）;CD4、CD69和RANTES、IL-5、IL-8的表达呈显著正相关（P〈0．01、P〈0．05和P〈0．05）。IL5、RANTES的高表达分别与嗜酸粒细胞浸润呈显著正相关（P〈0．05、P〈0．01）。IL8的表达和鼻息肉组织中微血管形成呈显著正相关（P〈0．01）。结论：CD4、CD69过度活化的T淋巴细胞及其产生的相关细胞因子IL5、IL-8、RANTES可能促进和形成鼻息肉的免疫发病过程;IL5、RANTES可促进嗜酸粒细胞的浸润、聚集、活化;IL-8可促进微血管形成。     

白细胞介素13对人鼻息肉黏膜上皮细胞嗜酸性趋化因子3蛋白含量及mRNA影响的实验研究

目的　观察白细胞介素13（interleukin-13,IL-13）刺激鼻息肉黏膜上皮细胞后嗜酸性趋化因子3,即CCL26蛋白含量及其mRNA表达变化,探讨嗜酸性粒细胞 浸润鼻黏膜的机制。方法　以IL-13细胞因子刺激原代培养的人嗜酸性鼻息肉黏膜上皮细胞。用实时定量PCR法检测CCL26 mRNA的表达,ELISA检测CCL26蛋白表达。结果  与对照组相比,IL-13刺激组鼻息肉黏膜上皮细胞CCL26蛋白分泌增加,差异具有统计学意义（P <0.05）。其增高呈浓度依赖性。结论　IL-13可诱导鼻息肉黏膜上皮细胞分泌CCL26蛋白。     

变应性鼻炎小鼠鼻黏膜中半乳糖凝集素3和9的表达

目的探讨小鼠鼻黏膜内半乳糖凝集素3（galectin-3）和半乳糖凝集素9（galectin-9）的表达水平及其与变应性鼻炎的关系。方法20只BALB／c小鼠随机分为变应性鼻炎组（AR组）和对照组，每组10只。AR组分别于第1天和第14天腹腔注射含卵清蛋白10μg及氢氧化铝2mg的生理盐水悬液0．1ml，第21天起鼻腔给予1％卵清蛋白局部激发，持续7d；对照组用生理盐水代替卵清蛋白。通过免疫组织化学染色和逆转录-聚合酶链反应（RT-PCR）检测鼻黏膜中galectin．3和galectin-9的表达，同时采用酶联免疫吸附测定法（ELISA）检测外周血中自细胞介素4（IL-4）、白细胞介素5（IL-5）及γ干扰素（IFN-γ）的表达。结果AR组和对照组均有galectin-3和galectin-9蛋白及mRNA的表达，AR组表达强度高于对照组，差异具有统计学意义（t值分别为11．18、9．261、7．667和6．971，P值均〈0．05）。与对照组相比，AR组小鼠外周血中IL-4和IL-5含量均有不同程度的增高（t值分别为7．202和8．121，p值均〈0．05），而IFN-γ含量则下降（t=-11．94，p〈0．05）。galectin3和galectin9mRNA的表达强度与外周血中IL-4、IL-5的含量呈正相关（r值分别为0．667、0．847、0．720及0．736，P值均〈0．05），与IFN-γ含量呈负相关（r值分别为-0．528和-0．817，P值均〈0．05）。结论galectin3和galectin9可能在变应性鼻炎的发病过程中发挥一定作用。     

基于高迁移率族蛋白B1/Toll样受体4信号途径研究慢性鼻-鼻窦炎伴鼻息肉发病过程中炎性因子的表达#br#

目的　探讨基于高迁移率族蛋白B1（HMGB1）/Toll样受体4（TLR4）信号研究慢性鼻-鼻窦炎伴鼻息肉（chronic rhinosinusitis with nasal polyp,CRSwNP）发病过程中炎性因子的表达。方法　HE检测CRSwNP中的嗜酸细胞;免疫组织化学、qRT-PCR和Western blot检测CRSwNP中HMGB1的表达;qRT-PCR检测CRSwNP中IL-4和IL-13的表达;ELISA检测HMGB1（HMGB1/TLR4通路）对人鼻黏膜上皮细胞中IL-4和IL-13表达的影响。结果  CRSwNP中嗜酸细胞浸润增加（P <0.001）,HMGB1表达上调（P <0.001）,IL-4 mRNA和IL-13 mRNA的表达增加（P 均<0.01）;HMGB1使人鼻黏膜上皮细胞中IL-4和IL-13的表达增加（P 均<0.001）;与HMGB1（100 ng/ml）孵育组比较,加入抗TLR4抗体孵育后人鼻黏膜上皮细胞中IL-4和IL-13的表达水平降低（P 均<0.001）。结论　CRSwNP组织中HMGB1高表达。脂多糖刺激人鼻黏膜上皮细胞中HMGB1的产生,进而通过TLR4上调人鼻黏膜上皮细胞中IL-4和IL-13的产生。     

白细胞介素-1β在过敏性鼻炎小鼠的表达及其对鼻上皮细胞肿瘤坏死因子α诱导蛋白2的影响

目的　探讨白细胞介素1β（IL-1β）在过敏性鼻炎（allergic rhinitis,AR）小鼠鼻黏膜的表达及其体外对鼻上皮细胞肿瘤坏死因子α诱导蛋白2（tumor necrosis factor-α-inducible protein-2,TNFAIP2）表达的影响。方法　建立AR小鼠模型,检测鼻黏膜IL-1β基因的表达;培养小鼠鼻原代上皮细胞,用不同剂量IL-1β刺激,检测上皮细胞中TNFAIP2基因和蛋白的表达。结果　AR小鼠鼻黏膜IL-1β基因表达相对值为8.7±1.74,显著高于对照组1.21±0.26（P =0.0017）;小鼠鼻上皮细胞经25、50 ng/ml IL-1β刺激后TNFAIP2基因表达的相对值分别为15.83±1.65、26.50±4.25,均显著高于对照组2.31±0.78（P <0.0001,P =0.0002）,且50 ng/ml刺激组显著高于25 ng/ml刺激组（P =0.04）;TNFAIP2蛋白表达呈相同趋势。结论　AR小鼠鼻黏膜IL-1β表达增强,IL-1β体外能够诱导鼻上皮细胞表达TNFAIP2,提示AR中IL-1β可能通过调节TNFAIP2的表达发挥作用。     

不同浓度SEB对兔上颌窦GATA-3及Th1/Th2细胞因子表达的影响

目的：探讨向兔上颌窦腔注入不同浓度金黄色葡萄球菌肠毒素B（SEB）对上颌窦黏膜组织中Th2特异性转录因子GATA-3及Th1与Th2细胞因子表达的影响。方法：48只兔随机分成low-dose SEB组和highdose SEB组,每组24只。造模后每天向low-dose SEB组和high-dose SEB组兔左上颌窦腔分别注入0.3μg/L SEB（0.6ng）及30μg/L SEB（60ng）各2ml。2组兔右上颌窦腔每天注入2ml生理盐水作为对照组。造模后3、7、14及28d每组随机选6只兔处死并获取双侧上颌窦黏膜标本进行检测。用ABC-ELISA法检测IFN-γ、IL-2、IL-4和IL-5的含量;采用Real-time PCR和免疫组织化学法检测GATA-3的表达。结果：在造模后7、14及28d,high-dose SEB组左上颌窦黏膜组织中IFN-γ和IL-2的水平均高于low-dose SEB组及对照组（均P〈0.05）。造模后14、28dlow-dose SEB组左上颌窦黏膜组织中IL-4和IL-5的水平均高于high-dose SEB组和对照组（均P〈0.05）。Real-time PCR结果显示,low-dose SEB组转录因子GATA-3mRNA的相对表达水平较high-dose SEB组和对照组均明显升高（均P〈0.05）。免疫组织化学结果显示,low-dose SEB组GATA-3表达的阳性细胞数高于high-dose SEB组和对照组（均P〈0.05）。结论：高浓度SEB促进Th1细胞因子分泌,而低浓度SEB增强Th2细胞因子表达及Th2型免疫反应。     

鼻息肉组织中固生蛋白C的表达与转化生长因子β1的关系

目的:探讨细胞外基质(extracellular matrix,ECM)成分固生蛋白C (tenascin C,TNC)在鼻息肉组织中异常表达的原因。方法:采用免疫组化方法检测TNC和转化生长因子β 1(transforming growth factor- β 1)在鼻息肉组织中的表达和关系。进一步采用细胞培养、实时定量RT-PCR和原位ELISA技术研究TGF- β1及嗜酸粒细胞对人呼吸道上皮细胞系BEAS-2B细胞TNC表达的调控作用。结果:①TNC和TGF- β 1蛋白在鼻息肉组织中的表达显著上调,TNC表达强度与TGF-β 1 阳性细胞总数(r=-0．58,P<0．01)和TGF-β 1阳性的嗜酸粒细胞数显著相关(r=-0．61,P<0．01);②浓度为1ng／ml和10ng／ml的 TGF-β 1刺激4h后BEAS-2B细胞TNC mRNA的表达分别为未刺激状态下的(7．20±3．43,x±s,下同)倍和(22．48±5．35)倍, 与未刺激状态下的表达水平相比差异有统计学意义(P值<0．01); 与刺激24h后的TNC蛋白表达的荧光强度相比差异亦有统计学意义(P <0．05);③BEAS-2B细胞和嗜酸粒细胞以2．1、1:1和1:2的数量比例进行共培养,4h后BEAS-2B细胞TNC mRNA的表达与未刺激状态下的表达水平相比,差异均有统计学意义(P值均<0．01);24h后 TNC蛋白表达的荧光强度与未刺激状态下的荧光强度相比差异亦均有统计学意义(P值均<0．05);嗜酸粒细胞的这种诱导作用可以被抗 TGF-β 1的中和抗体显著抑制(P<0．05)。结论:TGF-β 1和嗜酸粒细胞可以诱导呼吸道上皮细胞对TNC的表达,嗜酸粒细胞的作用部分通过TGF-β 1介导,鼻息肉组织中TNC表达的增高同嗜酸粒细胞来源的TGF-β 1有关。图9参16     

Hypoxia effects on vascular endothelial growth factor derived epithelial cells of nasal polyps]

OBJECTIVE: To understand the role of nasal mucous epithelial cells to hypoxia in early stage of nasal polyps(NP) formation. METHODS: Epithelial cells of NP and inferior turbinate (IT) were cultured without serum under normal oxygen and hypoxia, and stimulus of inflammatory cytokines. Erythropoietin (EPO) was regarded as hypoxia mark, and expression of vascular endothelial growth factor(VEGF) mRNA and protein derived from epithelial cells were detected respectively by in situ hybridization and ELISA. RESULTS: 1. Under hypoxia, EPO mRNA was expressed intensely in epithelial cells from NP and IT, and there was no significant difference between both of them. This result suggested that EPO might be regarded as a hypoxic mark. 2. The ability of producing VEGF mRNA increased with cytokines stimulation, especially under hypoxia. Protein level of VEGF from epithelial cells of NP and IT increased with cytokines stimulation, especially in hypoxia and was time-dependent. CONCLUSION: Epithelial cells actively produce vast VEGF under hypoxia. The VEGF induced by hypoxia of the mucosa in middle meatus is of importance in the formation of nasal polyps(NP) in early stage, which may be the major cause of NP formation in middle meatus.     

鼻息肉中免疫球蛋白E白细胞介素5和粒细胞巨噬细胞集落刺激因子的表达

目的 :探讨鼻息肉组织中免疫球蛋白E(IgE)、白细胞介素 5 (IL 5 )和粒细胞巨噬细胞集落刺激因子(GM CSF)的表达对嗜酸性粒细胞 (EOS)浸润聚集的作用及IgE与IL 5和GM CSF的关系及其意义。 方法 :应用连续两步免疫酶法 (Sandwich)检测 31例鼻息肉标本 (鼻息肉组 )和 11例下鼻甲黏膜标本 (对照组 )组织匀浆中IgE水平 ;应用酶联免疫吸附测定法检测两组组织匀浆中IL 5和GM CSF水平 ,同时观测两组组织中EOS的浸润程度。结果 :鼻息肉组IgE、IL 5和GM CSF水平均明显高于对照组 (圴 P <0 .0 1) ,且三者水平分别与鼻息肉组织中EOS浸润数呈正相关 (r分别为 0 .73、0 .5 4和 0 .4 8,均P <0 .0 1) ,且鼻息肉中IgE与IL 5和GM CSF水平呈正相关 (r分别为 0 .6 5和 0 .4 2 ,分别P <0 .0 1和P <0 .0 5 )。结论 :IgE局部水平的上调提示鼻息肉中存在局部变态反应 ,其对鼻息肉中IL 5和GM CSF的表达上调和EOS的聚集具有重要作用 ;IL 5和GM CSF在鼻息肉中高表达 ,对EOS的浸润聚集具有重要作用。     

Na^＋通道与鼻息肉形成关系的初步研究

目的 探讨Ｎａ＾＋通道与鼻息肉形成的关系,并进一步探讨白细胞介素１β（ＩＬ－１β）对鼻粘膜上皮细胞Ｎａ＾＋通道的影响。方法 采用细胞贴附式膜片钳技术对无血清气－液界面培养的鼻源性鼾症患者下鼻甲和鼻息肉上皮细胞进行研究。结果 ①比较与Ｎａ＾＋回吸收相关的３种因素,即Ｎａ＾＋通道的电导、开放概率（开放时间／采样时间）及发生率（Ｎａ＾＋通道的阳性膜片／检测膜片）,发现鼻息肉上皮细胞的电导和发生率均略高于     

Staphylococcal enterotoxin B induced expression of IL-17A in nasal epithelial cells and its association with pathogenesis of nasal polyposis

Interleukin (IL)-17A is a highly inflammatory cytokine and is known to be produced by Th17 cells. The importance of IL-17A expression in nasal epithelial cells is not well understood. The goal of this study is to explore the expression of IL-17A in nasal epithelial cells in vivo and in vitro. IL-17A and staphylococcal enterotoxin B (SEB) were detected by immunofluorescence (IF) in nasal epithelial cells of control mucosa (n = 10) and nasal polyps (n = 20). Expression of IL-17A, RORC, IL-6, and TGF-β1 was also measured by RT-PCR in the tissue of control nasal mucosa (n = 10) and nasal polyps (n = 20). IL-17A expression was evaluated in the human nasal epithelial cells after SEB stimulation. Finally, IL-17A expression was demonstrated by immunohistochemistry and IF following intranasal SEB instillation in mice. Expression of IL-17A in nasal epithelial cells was higher in nasal polyps compared to control mucosa. There was a significant correlation between IL-17A and SEB detection in nasal polyps using IF. SEB increased IL-17A expression in human nasal epithelial cells, and in epithelial cells of SEB instilled mice. In conclusion, SEB exposure of nasal epithelial cells induces the enhanced expression of IL-17A. SEB may be involved in pathogenesis of nasal polyps by enhancing IL-17A expression in epithelial cells in nasal polyps.     

Effects of TNF-alpha and IL-1 beta on mucin, lysozyme, IL-6 and IL-8 in passage-2 normal human nasal epithelial cells

Little is known about the regulatory effects of cytokines on various nasal secretions in normal human nasal epithelial cells. The aim of this study was to examine whether TNF-alpha, IL-1beta or their combination can increase the secretion of mucin as an indicator of mucous secretion, the secretion of lysozyme as an indicator of serous secretion and the secretion of IL-6 and IL-8 as important cytokines. In addition, we wanted to examine their message levels in normal human nasal epithelium. On day 12 of culture, passage-2 normal human nasal epithelial cells were treated with 10 ng/ml TNF-alpha, 10 ng/ml IL-1beta and combinations of both. Twenty-four hours later, the apical secretions were collected. A mixture of TNF-alpha and IL-1beta synergistically increased secretion of mucin, IL-6 and IL-8, but did not increase secretion of lysozyme. A combination of TNF-alpha and IL-1beta showed a questionable increase of MUC2 mRNA levels. TNF-alpha, IL-1beta and a combination of both all significantly increased MUC8 mRNA levels. Neither TNF-alpha, IL-1beta nor a combination of both increased MUC5AC, MUC5B and lysozyme mRNA levels. IL-1beta alone or a combination of TNF-alpha and IL-1beta comparably increased IL-6 and IL-8 mRNA levels slightly. In conclusion, a mixture of inflammatory mediators can synergistically increase secretion of mucin, IL-6 and IL-8 in human nasal epithelium. Accordingly, nasal secretions may be under the control of an inflammatory mediator network.     

鼻息肉中单核细胞趋化蛋白1和血管内皮生长因子的表达

目的明确单核细胞趋化蛋白1（monocyte chemotactic protein 1，MCP-1）和血管内皮生长因子（vascular endothelial growth factor，VEGF）在鼻息肉组织中的表达及其相关性，初步探讨MCP-1与鼻息肉发生的关系。方法取40例鼻息肉组织和25例下鼻甲组织，应用原位杂交和免疫组织化学等方法检测MCP-1和VEGF mRNA及蛋白质的表达。结果鼻息肉组织中MCP-1和VEGF mRNA及蛋白质的表达均高于对照组下鼻甲组织（P值均〈0．01）；鼻息肉组织中MCP-1和VEGF蛋白质的表达呈正相关（r=0．871，P〈0．05）。结论鼻息肉组织中MCP-1和VEGF表达增加，二者协同作用可能是鼻息肉形成的原因之一。     

Lipopolysaccharide decreases connexin 43 expression on nasal epithelial cells in vitro

Decreased connexin 43 (Cx43) expression as a result of application of lipopolysaccharide (LPS) may limit the diffusion of intercellular signaling molecules that is essential to the coordinated function of neighboring cells. Therefore, it may be related to a ciliary beating defect in nasal epithelial cells and result in accumulation of harmful substances.Gap junction intercellular communication (GJIC) is altered during inflammation in tracheal epithelial cells. Thus, we aimed to investigate whether LPS affects the expression of Cx43, the elementary protein composing the gap junction of nasal epithelial cells, in vitro.LPS (Pseudomonas aeruginosa serotype 10) was applied to epithelial cells obtained from nasal polyp for 24 h in vitro. As an inflammatory indicator, IL-8 secretion was measured using a commercially available ELISA kit. Cx43 protein was detected semi-quantitatively using Western blotting.The nasal epithelial cells constitutively secreted IL-8 at a concentration of 0.45+/-0.03 ng/microg protein. In the presence of 10(-2) mg/ml LPS, the concentration of IL-8 was significantly increased to 0.55+/-0.05 ng/microg protein (n=8). Expression of the Cx43:beta-actin ratio decreased in a time- and dose-dependent fashion (10(-3)-10(-1) mg/ml LPS).     

鼻息肉组织中白细胞介素—5和粒细胞巨噬细胞集落刺激因子水平测定及其意义

OBJECTIVE: To study the relationship between the concentration of interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulation factor (GM-CSF) in nasal polyps and its significance. METHODS: The concentration of IL-5, GM-CSF was measured by enzyme linked immunosorbent assay (ELISA) in a total of 38 cases, and the number of eosinophils were counted in the same tissues. RESULTS: The statistical analysis showed that the concentrations of IL-5 and GM-CSF have significant difference (P < 0.05) between the nasal polyps and the control group. There is a positive correlation between the quality of IL-5/GM-CSF and the extent of eosinophils (r = 0.75/0.71). CONCLUSION: This study demonstrated that there are inductive compartments, namely the microenvironment, in which immune-effector and inflammatory cell function is modulated in nasal polyps. A special discussion has been made of eosinophils and IL-5/GM-CSF in the formation of nasal polyps, and the effect of the IL-5/GM-CSF in the accumulation of eosinophils.