高表达Ang1基因的间充质干细胞株的建立及其肺内转化研究

高表达Ang1基因的间充质干细胞的建立及其肺内转化研究徐金富瞿介明李惠萍等同济大学附属上海市肺科医院复旦大学附属华东医院Highpromiseforangiopoietin-1(Ang1)inlunginjuryinterventionAng1decreasedinLPSinducedlunginjuryanti-inflammation:inhibitsendothelialpermeability,neutrophiladherenceandIL-8productionAnti-apoptosisroletoendothelialcellsThurstonG,RudgeJS,IoffeE,etal.Angiopoietin-1protectstheadultvasculatureagainstplasmaleakage.NatMed.2000,6:460-463.

DalyC,WongV,BurovaE,etal.Angiopoietin-1modulatesendothelialcellfunctionandgeneexpressionviathetranscriptionfactorFKHR(FOXO1).GeneDev.2004,18(9):1060-1071.

PizurkiL,ZhouZ,GlynosK,etal.Angiopoietin-1inhibitsendothelialpermeability,neutrophiladherenceandIL-8production.BritJPharmacol.2003,139(2):329-336.Mesenchymalstemcells(MSC)NoethicaldifficultiesharvestMSCfromtheindividualtobetreatedNorejectionproblemsSelfmigratetowheretheinjuryislocatedRojasM,XuJ,WoodsCR,etal.Bonemarrow-derivedmesenchymalstemcellsinrepairoftheinjuredlung.AmJRespirCellMolBiol2005;33:145–152

RobertJD，AnnemarieBM．mesenchymalstemcells：biologyandpotentialclinicaluses.ExperimentalHematology,2000,28(8):87

Ourhypothesis+Ang1MSCsByLentiviralvectorMSCstransducedwithAng1MiceLPSinducedlunginjuryInvivointerventionMethodsandresultspCR2.1-Ang1:Prof.FedericoBussolinoinInstituteforcancerresearchandtreatment,ItalySequencingresults:100%coincidewithAng1cDNAsequenceElectrophoresisaftercuttingbyrestrictionenzymepLVpCMV-dR8.74pMD2GInvitroLentivirus-mediatedAng1/eGFPtransferintoMSCsIsolation,purificationandexpansionofMSCsBonemarrowwasharvestedfrom6weeksoldC57BL/6byflushingthefemursandtibiasSeparateMSCsfrombonemarrowbydensitygradientcentrifugationinFicoll(Pharmacia)CulturedandpassagedwithPeister’smethodPeisterA,MelladJA,LarsonBL,HallBM,GibsonLF,ProckopDJ.Adultstemcellsfrombonemarrow(MSCs)isolatedfromdifferentstrainsofinbredmicevaryinsurfaceepitopes,ratesofproliferation,anddifferentiationpotential.Blood2004;103:1662-1668.MSCstransducedwithLenti-Ang1/eGFP1×106

MSCs

frompassage4wereseededintoa12-wellplateMultiplicityofinfection(MOI):10，20，40，80MergephotosAng1mRNAinMSCsMMSCs

MSCs-Ang1Ang1proteininMSCsLPSinducedALI3mgpermlLPS:

Ecoli.Serotype055:B5Byaerosolizationfor20minusingaflowrateof6liters/min7successivedays

IngenitoEP,MoraR,CullivanM,MarzanY,HaleyK,MarkL,andSonnaLA.Decreasedsurfactantprotein-Bexpressionandsurfactantdysfunctioninamurinemodelofacutelunginjury.AmJRespirCellMolBiol2001;25:35-44

DeliverMSCstomiceByjugularveininjection1×106cellsin200µlNSpermouseSacrificemiceandcollectsamplesonday0,3,7,14afterinjectionAng1proteininlungs*QuantitationofAng1protein*GFPwasobservedinlungbyfluorescentmicroscopeFluores