简介：AIM:ToinvestigatetheeffectofhepatitisBvirus(HBV)XgeneonapoptosisandexpressionsofapoptosisfactorsinXgene-transfectedHepG2cells.METHODS:TheHBVXgeneeukaryonexpressionvectorpcDNVA3-XwastransientlytransfectedintoHepG2cellsbylipid-mediatransfection.UntransfectedHepG2andHepG2transfectedwithpcDNA3wereusedascontrols.ExpressionofHBxinHepG2wasidentifiedbyPT-PCR.MTTandTUNELwereemployedtomeasureproliferationandapoptosisofcellsin.threegroups.Semi-quantifiedRT-PCRwasusedtoevaluatetheexpressionlevelsofFas/FasL,Bax/Bcl-xL,andc-mycineachgroup.RESULTS:HBVXgenewastransfectedintoHepG2cellssuccessfully.RT-PCRshowedthatHBxwasonlyexpressedinHepG2/pcDNA3-Xcells,butnotexpressedinHepG2andHepG2/pcDNA3cells.AnalyzedbyMTT,cellproliferationcapacitywasobviouslylowerinHepG2/pcDNA3-Xcells(0.08910±0.003164)thaninHepG2(0.14410±0.004927)andHepG2/pcDNA3cells(0.12150±0.007159)(P＜0.05andP＜0.01).AnalyzedbyTUNEL,cellapoptosiswasmuchmoreinHepG2/pcDNA3-Xcells(980/2000)thanHepG2(420/2000),HepG2/pcDNA3cells(520/2000)(P＜0.05andP＜0.01).Evaluatedbysemi-quantifiedRT-PCR,theexpressionlevelofFas/FasLwassignificantlyhigherinHepG2cellstransfectedwithHBxthaninHepG2andHepG2/pcDNA3cells(P＜0.05andP＜0.01).Bax/Bcl-xLexpressionlevelwasalsoelevatedinHepG2/pcDNA3-Xcells(P＜0.05andP＜0.01).Expressionofc-mycwasmarkedlyhigherinHepG2/pcDNA3-XcellsthaninHepG2andHepG2/pcDNA3cells(P＜0.05andP＜0.01).CONCLUSION:HBVXgenecanimpaircellproliferationcapacity,improvecellapoptosis,andupregulateexpressionofapoptosisfactors.TheinterventionofHBVXgeneontheexpressionofapoptosisfactorsmaybeapossiblemechanismresponsibleforthechangeincellapoptosisandproliferation.

简介：瞄准：调查在肝的卵形的房间在试管内的增长和区别上表明小径的正规Wnt的激活的效果。方法：WB-F344房间与recombinantWnt3a被对待(20，40，80，160，200ng/mL)在24h的没有浆液的媒介。房间增长被Brdu加入分析测量；未经治疗的WB-F344房间作为控制被拿。有为24h的Wnt3a(160ng/mL)的术后疗法，在对待的WB-F344房间代替细胞的本地化和beta-catenin的蛋白质表示并且与Wnt3a未经治疗被免疫荧光染色和西方的污点分析检验。CyclinD1mRNA表示被半量的反向抄本的聚合酶链反应(RT-PCR)决定。一些表型的标记的mRNA层次(法新社，CK-19，白长袍的)并且二个肝的原子因素(HNF-4，HNF-6)被RT-PCR测量。CK-19和法新社蛋白质的表情被西方的污点分析检测。结果：Wnt3a支持了WB-F344房间的增长。有recombinantWnt3a的WB-F344房间的刺激导致了transcriptional使活跃之物beta-catenin的累积，和它进原子核，和起来调整的典型Wnt目标基因CyclinD1的易位。在当浆液不在时的Wnt3a处理的3d以后，WB-F344房间保留了他们的双性人潜力表示hepatocytes和cholangiocytes的几个特定的表型的标记，例如法新社和CK-19，跟随表明小径的正规Wnt的激活。结论：表明小径的正规Wnt支持老鼠的增长和自强肝的卵形的房间。

简介：AIMToinvestigatetheroleofthecomplement5a(C5a)/C5areceptor(C5aR)pathwayinthepathogenesisofacuteliverfailure(ALF)inamousemodel.METHODSBALB/cmicewererandomlyassignedtodifferentgroups,andintraperitonealinjectionsoflipopolysaccharide(LPS)/D-galactosamine(D-GalN)(600mg/kgand10μg/kg)wereusedtoinduceALF.TheKaplanMeiermethodwasusedforsurvivalanalysis.Serumalanineaminotransferase(ALT)levels,atdifferenttimepointswithina1-wkperiod,weredetectedwithabiochemistryanalyzer.Pathologicalexaminationoflivertissuewasperformed36hafterALFinduction.Serumcomplement5(C5),C5a,tumornecrosisfactor-α(TNF-α),interleukin(IL)-1β,IL-6,high-mobilitygroupproteinB1(HMGB1)andsphingosine-1-phosphatelevelsweredetectedbyenzyme-linkedimmunosorbantassay.Hepaticmorphologicalchangesat36hafterALFinductionwereassessedbyhematoxylinandeosinstaining.ExpressionofC5aR,sphingosinekinase1(SphK1),p38-MAPKandp-p38-MAPKinlivertissue,peripheralbloodmononuclearcells(PBMCs)andperitonealexudativemacrophages(PEMs)ofmiceorRAW264.7cellswasanalyzedbywesternblotting.C5aRmRNAlevelsweredetectedbyquantitativereal-timePCR.RESULTSActivationofC5andup-regulationofC5aRwereobservedinlivertissueandPBMCsofmicewithALF.BlockadeofC5aRwithaC5aRantagonist(C5aRaC5aRa)significantlyreducedthelevelsofserumALT,inflammatorycytokines(TNF-α,IL-1βandIL-6)andHMGB1,aswellasthelivertissuedamage,butincreasedthesurvivalrates(P<0.01forall).BlockadeofC5aRdecreasedSphK1expressioninbothlivertissueandPBMCssignificantlyat0.5hafterALFinduction.C5aRapretreatmentsignificantlydownregulatedthephosphorylationofp38-MAPKinlivertissuesofALFmiceandC5astimulatedPEMsorRAW264.7cells.Moreover,inhibitionofp38-MAPKactivitywithSB203580reducedSphK1proteinproductionsignificantlyinPEMsafterC5astimulation.CONCLUSIONTheC5a/C5aRpath

简介：AIMToexploretheprotectiveeffectsandunderlyingmechanismsoftotalpolysaccharidesoftheSijunzidecoction(TPSJ)ontheepithelialbarriersinvitro.METHODSCaco-2cellmonolayersweretreatedwithorwithoutTPSJinthepresenceorabsenceofTNF-α,andparacellularpermeabilityandtransepithelialelectricalresistance(TEER)weremeasuredtoevaluatetheepithelialbarrierfunction.Immunofluorescenceandwesternblottingwererespectivelyusedtoevaluatethedistributionandexpressionofthetightjunctionproteinsclaudin1,claudin2,zo3,andoccludininCaco-2cells.Westernblottingwasalsousedtoevaluatethecellularexpressionofmyosinlightchain(MLC),phosphorylatedMLC(pMLC),MLCkinase(MLCK),andnuclearfactor(NF)-κBp65.RESULTSTPSJpromotedtheproliferationofCaco-2cellsandinhibitedTNF-α-inducedsecretionofpro-inflammatorycytokines.Furthermore,TPSJsignificantlyamelioratedboththereductionofTEERandtheincreasedparacellularpermeabilityobservedintumornecrosisfactor(TNF)-α-damagedCaco-2monolayers.Furthermore,TPSJremarkablyattenuatedTNF-α-inducedmorphologicalchanges,downregulatedtheexpressionofclaudin1,claudin2,zo3,andoccludin,andmarkedlysuppressedTNF-α-mediatedupregulationofp-MLCandMLCKexpression.Finally,TPSJinhibitedtheactivationandexpressionofNF-κBp65.CONCLUSIONOurresultsdemonstratethatTPSJalleviatestheTNF-α-inducedimpairmentoftheintestinalepithelialcellbarrierfunctionbysuppressingNF-κBp65-mediatedphosphorylationofMLCKandMLC.