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雷帕霉素对TGF-β2诱导的HLECs增殖、迁移及上皮间质转化的影响.docx

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雷帕霉素对TGF-β2诱导的HLECs增殖、迁移及上皮间质转化的影响 Introduction Posteriorcapsuleopacification(PCO)isoneofthemostcommoncomplicationsofcataractsurgery,whichaffectsthevisualacuityofpatients.Humanlensepithelialcells(HLECs)aretheprimarycellsresponsiblefortheformationofPCO,whichischaracterizedbytheproliferation,migration,andfibrosisofHLECs.Transforminggrowthfactor-beta2(TGF-β2)isacrucialcytokineintheprocessofPCO,whichcaninducetheproliferation,migration,andepithelial-mesenchymaltransition(EMT)ofHLECs.Therefore,theinhibitionofTGF-β2-inducedHLECproliferation,migration,andEMTisapotentialstrategyforpreventingandtreatingPCO.ReparixinisaselectiveinhibitoroftheN-formylpeptidereceptor1(FPR1),whichcaninhibittheproliferationandmigrationofvariouscells.Inthisstudy,weinvestigatedtheeffectsofreparixinonTGF-β2-inducedHLECproliferation,migration,andEMT. MaterialsandMethods Cellculture:HLECswereobtainedfromthecapsularbagsofpatientswhounderwentcataractsurgeryandwereculturedinDulbecco'smodifiedEagle'smedium(DMEM)supplementedwith10%fetalbovineserum(FBS)and1%penicillin/streptomycin. Cellviabilityassay:TheeffectsofreparixinandTGF-β2onHLECviabilityweredeterminedbytheMTTassay.HLECswereseededin96-wellplatesandtreatedwithvariousconcentrationsofreparixin(0-10μM)andTGF-β2(5ng/ml)for24h.Then,20μlofMTTsolutionwasaddedtoeachwell,andthecellswereincubatedforanother4h.Afterremovingthemedium,150μlofDMSOwasaddedtodissolvetheformazancrystals,andtheabsorbancewasmeasuredat490nmusingamicroplatereader. Cellmigrationassay:HLECmigrationwasassessedusingaTranswellassay.HLECswereseededintheupperchamberoftheTranswellinserts(8μmporesize),andthelowerchamberwasfilledwithDMEMcontaining10%FBSand5ng/mlTGF-β2,withorwithoutreparixin.After24hofincubation,themigratedcellswerefixedwith4%paraformaldehydeandstainedwithcrystalviolet.Thecellsinfiverandomlychosenfieldswerecountedunderamicroscope. Westernblotanalysis:HLECsweretreatedwithTGF-β2(5ng/ml)andreparixin(5μM)forvarioustimeperiods.Thetotalproteinwasextracted,andtheexpressionofEMTmarkers(E-cadh