人外周血巨噬细胞培养及功能鉴定-林炜明

doi10．3969/j．issn．1000-484X．2015．01．018①②364000Ｒ292.12A1000-484X201501-0086-04①JKNo.JK2011053、2013N0026。②364000。[e-mail]。PBMC。CD14+10%AB10ng/mlM-CSFIMDMCD14+。CD14+CD1410%CD1485.8%。740～45μmＲaji。CD14+。CultureandfunctionalidentificationofmacrophagesfromhumanperipheralbloodLINWei-MingDAIAi-LingYinHui-FangYANGXiao-Yan.CollegeofLifeSciencesofLongyanUniversityLongyan364000ChinaAbstractObjectiveToisolatemonocytesfromhumanperipheralbloodmononuclearcellsPBMCinducemacrophagesandidentifythefunctionofmacrophages.MethodsMonocyteswereisolatedfromPBMCusingmagneticactivatedcellsortingMACSanti-CD14microbead.SortedCD14+andCD14－cellswerecheckedbyflowcytometertoevaluatetheefficiencyofsorting.ThesortedCD14+cellswereculturedinIMDMmediawith10%humanABserumand10ng/mlM-CSFfor7daystogeneratemacrophageswhichwereidentifiedbymorphologicalfeaturesandphagocytosisfunction.ＲesultsAhighpurityofmonocyteswasobtainedbyMACSanti-CD14microbead.ThepercentageofCD14+cellswas10%and85.8%beforeandaftersortingrespectively.Themacrophageswereapproximately40-45μminmaximumdiameterandhadthefriedeggcolonymorphologicalfeaturesafter7daysculture.ThelymphomaＲajicellswereefficientlyengulfedbymacrophages.ConclusionThehighpurityofCD14+monocytesisisolatedfromPBMCandmonocyte-derivedmacrophagesefficientlyengulfedlymphomacells.KeywordsMacrophagesMagneticactivatedcellsortingCultureFunctionalidentification-。。1。M-CSF2。CD14、、CD1434。CD14+。11.1IMDM、、ABTrypLELifeTechnologyM-CSFeBiosciencesPrimocinInvivogenCD14CD14cloneMoP9APCEasySepStemcellTechnol-ogies。682015311.2FicollPBMC2%FBSPBSPBMCPBMCDNaseⅠ15min30μm2.0×108PBMCCD14200μlEasySep15min100μlEasySep10min5min。2。1.3PBMC、CD14+5×1052.5μlCD14-APCcloneMoP9IgG4℃30minCD14CD14。1.410%AB10ng/mlM-CSFIMDM8ml10cmCD14+5×10637℃、5%CO27d。1、3、6、、。1.5Ｒaji1.5.1CFSEＲaji1.0×106Ｒaji0.5μmol/LCFSEPBS2minFBSFBS10%37℃10minCFSEＲaji。1.5.27PBSTrypLE15～20min1.0×106/ml。CFSEＲaji2.0×106/ml。50μl1∶29637℃、5%CO22h1μlCD14-APC4℃30min。22.11CD14PBMC10%CD14CD1485.8%CD14CD140.3%。CD14+。2.2CD14+CD14+10%AB10ng/mlM-CSFIMDM7d24h2A。31CD14Fig．1FlowcytometrygraphofpurityanalysisofCD14positivecellsNoteCD14expressionwasquantifiedflowcytometricanalysisusingCD14-APCantibodycloneMoP9.Blacklinesrepresentisotypecontrols.ＲedlinesrepresentA.PBMCbeforesortingB.NegativelyselectedcellsC.Positivelyselectedcells.7812Fig．2MorphologicalfeaturesofmacrophagesNoteMacrophagesculturedfor1dayA3daysB6daysC.3Fig．3FlowcytometrygraphofphagocytosisfunctionofmacrophageNoteA.MacrophagesbeforephagocytosisB.Macrophagesafterphago-cytosis2B。640～45μm2C。2.37CD14CD14+46%。56。CFSEＲaji2hCD14-APC3。82%CD1483%CD14+Ｒaji。3-、2。3。7。CD14CD14CD1410%85.8%。CD14CD14BA-8CD14+MoP9CD14CD14CD14。GM-CSF、IL-48M-CSF2M-CSFＲOSSHP1PI3K/AKT/9。10。10%AB10ng/mlM-CSF88201531。76。CD14+7dTrypLETrypsin15～20minTrypLETrypsin。7CD14CD14CD14。5。Erbel4CD14CD45ＲO。。ＲajiＲaji83%CD14+Ｒaji。11。ＲajiCD47FcCD47Fc12。CD14+、。1.-J．2014301126-132.2GeissmannFManzMGJungSetal．DevelopmentofmonocytesmacrophagesanddendriticcellsJ．Science20103275966656-661.3ChengSMChangSJTsaiTNetal．Differentialexpressionofdis-tinctsurfacemarkersinearlyendothelialprogenitorcellsandmon-ocyte-derivedmocrophagesJ．GeneExpr201316115-24.4ErbelCＲuppGHelmesCMetal．Aninvitromodeltostudyhet-erogeneityofhumanmacrophagedifferentiationandpolarizationJ．JVisExp201376e50332.5KangJWChoiSCChoMCetal．Aproinflammatorycytokinein-terleukin-32βpromotestheproductionofananti-inflammatorycy-tokineinterleukin-10J．Immunology20091281e532-540.6.DC-STAMPJ．2013295474-480.7.J．201329188-92.8DelirezhNMajediLＲezaeiSAetal．Generationofmaturemono-cyte-deriveddendriticcellsinthepresenceofheparinandmono-cyteconditionedmediumphenotypicandfunctionalcomparisonJ．IranBiomedJ201115379-84.9ChoiHKKimTHJhonGetal．ＲeactiveoxygenspeciesregulateM-CSF-inducedmoncyte/macophageproliferationthroughSHP1oxidationJ．CellSignal201123101633-1639.10HodreaJDeményMAMajaiGetal．Transglutaminase2isex-pressedandactiveonthesurfaceofhumanmonocye-derivedden-driticcellsandmacrophagesJ．ImmunolLett2010130174-81.11.J．2011322197-203.12ChaoMPAlizadehAATangCetal．Anti-CD47antibodysyner-gizeswithrituximabtopromotephagocytosisanderadicatenon-hodgkinlymphomaJ．Cell20101425699-713.2014-08-202014-09-10981