简介:Objective:Toinvestigatethebiologicalfunctionofplatelet-derivedgrowthfactorB(PDGF-B)onthesurvivalandproliferationofcatcornealendothelialcellssoastoprovidebasesforfurtherstudiesofitsroleinwoundrepairanditsclinicalapplication.Methods:TotalRNAwasextractedfromtheplacentatissuesofhealthypregnantwomenundergoinghysterotokotomyandPDGFeDNAwasobtainedwithre-versetranscription-polymerasechainreaction(RT-PCR).TheprokaryoticexpressionvectorpET-PDGF-BwasconstructedandexpressedtherecombinantPDGF-BinEscherichiacoli(E.coli)BL21(DE3).AfterpurificationandrefoldingonNi2+-chelationaffinitychromatography(NTA)column,itwasusedtoculturecatcornealendothelialcells.Cellproliferationwastestedbymodifiedtertrazoliumsalt(MTT)andflowcytometer.Andthemorphologicchangeandtheultrastructurewereob-servedunderaninvertedphasecontrastmicroscope,ascan-ningelectronmicroscopeandatransmissionelectonmicroscope,respectively.Results:PDGF-Bchainpeptide(PDGF-BB)genewassuccessfullyinsertedintotheprokaryoticexpressionvector,pET-28a(+).ThepurifiedrecombinedproteinpET-PDGF-Bshowedasinglebandonsodiumdodecylsulfatepolyacry-lamidegelelectropheresis(SDS-PAGE)withthemolecularweightofabout27u,whichwasinagreementwiththede-ducedvalue.MTTandflowcytometryshowedthatPDGF-BBpromotedthesurvivalandproliferationofcatcornealen-dothelialcells.Conclusions:Theconstructionofrecombinantprokary-oticexpressionvectorpET-PDGF-BandthepreparationofPDGF-BBproteinprovideafoundationforfurtherstudyofthefunctionofPDGF-BBandproducingbiologicalPDGF-BBprotein.TheexpressedPDGF-BBpromotestheprolif-erationofculturedcatcornealendothelialcells.