华中科技大学硕士学位论文p120在机械划伤致气道上皮细胞炎症反应中对NF-κB信号通路的影响姓名:秦灵芝申请学位级别:硕士专业:病理学与病理生理学指导教师:王曦2010-12华中科技大学硕士学位论文2p120在机械划伤致气道上皮细胞炎症反应中对NF-κB信号通路的影响中文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信号转导途径的华中科技大学硕士学位论文3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﹤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。华中科技大学硕士学位论文4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。关键词人气道上皮细胞;p120;NF-κB;IκBα;IL-8;炎症反应华中科技大学硕士学位论文5Effectofp120onNF-κBsignalingpathwayduringtheairwayepithelialinflammationinducedbymechanicalscratchingAbstractBackgroundAirwayconsistingoftracheaandbronchiisthefirstdefensetoresistharmfulstimuli.Mechanicalscratchingasthemainstimulusinvitromodelmaycauseairwayinjury,resultinginpolarization,necrosisandsecretionofinflammatorycytokinesofthebronchialepithelialcells.Thisprocessrepeatingpersistentlycandevelopintochronicairwayinflammation,whichisthebasisofvariouslungdiseases,suchasCOPD,chronicpulmonaryfibrosisandlungcancer.Numerousstudiesindicatethattheactivationofnuclearfactor-κB(NF-κB)signalingpathwayisinvolvedinthecellgrowth,proliferation,apoptosisandinflammatorygenetranscription,etc.Itisthecoreofinflammatoryresponses,andcloselyassociatedwithmechanicalscratchinginducedairwayinflammation.Howevertheexactmechanismisstillunknown.InhibitorofNF-κB(IκB),amasterswitchforNF-κBactivating,usuallybindsNF-κBdimmerintoNF-κB/IκBcomplexinrestingcells,andthiscytoplasmicNF-κBisinactive.WithexternalstimulisuchasmechanicalscratchinginducingtheactivationofIκB,NF-κBimmediatelybecomesreleasedandactivated.Ittranslocatesintonucleusandbindswithtargetgenes,forexampleIL-8,thusenhancinginflammatorygenetranscription.p120-catenin(p120),belongstoanewmemberoftheArmadilloproteinfamily,whichisidentifiedwhenscreeningasubstrateforoncogenicSrcfamilytyrosinekinase.Previousstudiesfocusedonp120-mediatedcelladhesionandtumorgenesis.Recentstudiesrevealedthatp120isassociatedwithskin,salivaryglandsinflammationandinflammatoryboweldiseasesthroughinvokingtheNF-κBsignalingpathway.Buttheresearchontherelationshipofp120andtheairway华中科技大学硕士学位论文6inflammationisrare.Thereforeourstudiesfocusedonchangesofp120anditseffectsonNF-κBsignalingpathwayduringtheairwayinflammationinducedbymechanicalscratching.ObjectiveUsingawidelyusedinvitromodelbyscratchinghumanbronchialepithelialcells(HBECs),weinvestigatetheexpressionchangesofp120anditseffectsonNF-κBsignalingpathway.Thisstudymaybringtheoreticalandexperimentalevidencesforfurtherexploringthemechanismofairwayinflammation.MethodsInthemodelofairwayinflammationbyscratchingamonolayerofHBECsinvitro,weutilizedWesternblottodetecttheexpressionsofp120,NF-κBp65andIκBα.Next,weexaminedNF-κBp65translocationintonucleusbyisolationofcytoplasmicandnuclearproteins.Finally,wedetectedtheexpressionofIL-8byenzyme-linkedimmunosorbentassay(ELISA).ResultsByWesternblot,weexaminedtheexpressionofp120isoformsinHBECs,mainlycomposedofisoform1(120KD)andisoform3(100KD).Andtheexpressionofisoform1wasweak.Thenwefoundthatscratchingdecreasedp120andIκBα,butsignificantlyincreasedtheamountofNF-κBp65atdifferenttimepoints.ItindicatesthatthephosphorylationanddegradationofIκBαactivatedNF-κB.Moreover,thedecreasingofp120activatedNF-κBsignalingpathway.IsolationofcytoplasmicandnuclearproteinsshowedNF-κBp65translocationintonucleus.Meanwhile,thenuclearexpressionofNF-κBp65wasgraduallyincreasedinatime-dependentmannerafterscratching.FinallyELISAdetectedtheincreasedproductionofIL-8(p﹤0.05).Conclusions1Mechanicalscratchinginhibitstheexpressionofp120inHBECs,andactivatesNF-κBsignalingpathway.2PhosphorylateddegradationofIκBαinvolvesintheactivationofNF-κB华中科技大学硕士学位论文7signalingpathwayaftermechanicalscratchingHBECs.3AfteractivationofNF-κBsignalingp