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CD107aNK龚春香1,2,王长荣1,龚卫娟1,胡茂志3,季明春1(1.,,225001;2.,,213200;3.,,225001) : CD107a、NK。 (PBM-Cs)K5623∶1,2h,1.5hCD107aCD56,CD107a。CD107a、CD107a。LDHNK。 NKCD107a,CD107a,。LDH。 CD107aNK、、。:CD107a;NK;:R392.33 :A :1672-2353(2010)13-0023-04DetectionofNKcellcytotoxicitywithCD107aantibodylabelingbyFlowCytometryGONGChun-xiang1,2,WANGChang-rong1,GONGWei-juan1,HUMao-zhi3,JIMing-chun1(1.DepartmentofImmunology,MedicalCollegeofYangzhouUniversity,Yangzhou,Jiangsu,225001;2.Departmentofpathology,JintanPeople′sHospital,Jintan,Jiangsu,213200;3.TestingCentreofYangzhouUniversity,Yangzhou,Jiangsu,225001) ABSTRACT:Objective TodetectNKcellcytotoxicitybyflowcytometryfollowingCD107aantibodylabeling.Methods Atfirstperipheralbloodmononuclearcells(PBMC)wereisolatedandincubatedwithK562targetcellsataratioof3∶1.Twohourslater,monensinwasaddedintotheculturesystem.After1.5hoursCD107aandCD56doublepositivecellswerelabeledwiththeircorre-spondingantibodies,andanalyzedbyflowcytometry.TheeffectsondetectingsensitivitybyCD107aantibodyincubationtimeandtheratiosofPBMCandK562cellswerealsoexamined.LastlywecomparedthismethodwiththeconventionalLDHreleasingmethod.Results CD107apositivecellscouldbeeasilydetectedonNKcellsfollowingstimulation.IfCD107aantibodywasaddedintoculturemediumafterPBMCandK562cellsincubation,non-specificlabelingwassignificantlydecreased.ThedetectingresultbythismethodwasinconformityconfirmativetothatbytheLDHreleasingmethod.Conclusions ThemethodwedevelopedherefordetectingcytotoxicityofNKcellswasrapid,sensitiveandneededasmallamountofeffectors.KEY WORDS:CD107a;naturalkillercells;cytotoxicity NK,、。,、[1]。-1(LAMP-1CD107a),50%[2-3]。NK,,,[4]。:2010-03-20:(30671917);(BK2004404,BK2008215)20101413JournalofClinicalMedicineinPractice·23·,CD107a,CD107a[5]。,CD107aNKNK。,51Cr,51Cr[6]。(LDH),[7],、、、、。EGFPK562,(PI),PIEGFPEGFP,NK[8]。CD107aNK,,CD107aNK。1 1.1 实验材料Axis-shield。CD56-FITC(MEM188),CD107a-PECy5(eBioH4A3)eBioscience。(PMA)、Sigma。RPMI-1640invitrogen,。LDHCytoTox96non-ra-dioactivecytotoxicityassayPromega。1.2 方法1.2.1 :K562,MHCⅠ。10%RPMI1640,37℃,5%CO2。1.2.2 PBMCs:PBS,Ficoll2∶1Ficoll,2000rpm,15min,,。,PBS2,RPMI-1640,。1.2.3 (FCM)NKCD107a:RPMI1640PBMCs,1×106/mL,K562。PBMCsK562,U,37℃CO22h,(mon-ensin,2μmol/L)3.5hPE-Cy5-CD107a、FITC-CD56h。PBS3,200μL1%。PBMCs(PMA,2.5μg/mL)(Ionomycin,0.5μg/mL)。1.2.4 LDHNK:PBMCsK5621:3、1:1、3:1,250g4min,37℃5%CO24h,,30min,490nm。、、、、,4。=(--)/(-)×100。2 2.1 FCM分析CD56和CD107a双阳性细胞(PBMC)K5623∶1,FITC-CD56PE-Cy5.5-CD107a,FCMNK。,NK,CD107a,PMA/Ionomycin。,(Q2/Q2+Q4)×100CD107aNK(%)。NK=CD107a(%)-CD107a(%)。2.2 CD107a抗体孵育时间对FCM检测结果的影响CD107a,Alter。,[9]。CD107aPBMCsK562,NK()CD107a,。PBMC4hNKCD107a,PBMC()CD107a,1h·24·141 FCMCD56+CD107a+,4hCD56FCM,PBMC。CD107a4h,CD107a(2),。2 MonensinCD107a ,Alter,2:1PBMCsK562CD107a,2h,2hCD56;PBMCsK562,2h,1.5hCD107aCD56。,1NKCD107aK562CD56+CD107a+,2NKCD107a,CD56+CD107a+CD107a8(3)。3 CD107aFCM2.3 不同效靶比例对FCM检测结果的影响PBMCsK562,2.5∶1、5∶1、10∶1、20∶1、40∶122,,CD56+CD107a+(4)。PBMCsK5623∶1、1∶11∶3,,CD56+CD107a+(5),,。2.4 CD107a检测法与传统LDH释放法的关联LDH,,CD107aLDHNK。,1∶3、1∶1、3∶1,2,CD107a·25·13:CD107aNK(6),LDH。4 CD107NK5 CD107NK6 CD107aLDH3 NK、,CD107a,CD107aNKNK,NK。,、LDH,CD107a,CD107a。,CD107aNK,、、。NKCD107a,[10],NKCD107a,1.2%～5.8%。,NKCD107a,NK。NK,CD107a[11],NK,CD107a,。NK,NK,CD107a(2),Q1、Q2CD107a。CD107a,。AlterNKCD107a,CD107a,。,2h,NK,NK,CD107a,CD107a,CD107ah,。3CD107a,Q1;CD107a,Q1(CD56-)。51CrLDH,,。,,,。CD107a(NK),,,NK。,,CD107aNK(4,5)。,CD107a(下转第31面)·26·14,。,,。[1] ,,,.[J].,2000,5(4):421.[2] JiaoK,LiuH,ChenJ,etal.Rolesofplasmainterleukin-6andtumornecrosisfactor-aandFFAandTGinthedevelop-mentofinsulinresistanceinducedbyhigh-fatdiet[J].Cy-tokine,2008,42(2):161.[3] XuH,BarnesGT,YangQ,etal.Chronicinflammationinfatplaysacrucialroleinthedevelopmentofobesity-relatedinsulinresistance[J].JClinInvest,2003,112(12):1821.[4] DespresJP,LemieuxI.Abdominalobesityandmetabolicsyndrome[J].Nature,2006,444(7121):881.[5] DonathMY,SchumannDM,FaulenbachM,etal.Isletin-flammationintype2diabetes:frommetabolicstresstothera-py[J].DiabetesCare,2008,31(Suppl.2):S161.[6] SauterNS,SchulthessFT,GalassoR,etal.Theanti-inflam-matorycytokineIL-1Raprotectsfromhighfatdiet-inducedhyperglycemia[J].Endocrinology,2008,149(5):2208.[7] ,.[J].,2004;10(1):144.[8] StewartLK,WangZ,RibnickyD,etal.Failureofdietaryquercetintoalterthetemporalprogressionofinsulinresis-tanceamongtissuesofC57BL/6Jmiceduringthedevelop-mentofdiet-inducedobesity[J].Diabetologia,2009,52(3):514.[9] GastaldelliA,CusiK,PettitiM,etal.Relationshipbetweenhepatic/visceralfatandhepaticinsulinresistanceinnondia-beticandType2diabeticsubjects[J].Gastroenterology,2007,133(2):496.[10] GustafsonB,SmithU.CytokinespromoteWntsignalingandinflammationandimpairthenormaldifferentiationandlipidaccumulationin3T3-L1preadipocytes[J].JBiolChem,2006,281(4):9507.[11] PiconiL,QuagliaroL,CerielloA.Oxidativestressindia-betes.ClinChemLabMed,2003,41(9):1144.[12] CerielloA,TestaR.Antioxidantanti-inflammatorytreat-mentintype2diabetes[J].DiabetesCare,2009,32(Suppl.2):S232.(上接第26面),。,CD107a[4],NK。[1] CooperMA,FehnigerTA,CaligiuriMA.Thebiologyofhu-mannaturalkillercellsubse