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一、抗原肽诱导CTL制备
1.PBMCswereseparatedfromthewholebloodofHLA-A2+healthycontrols.
6
PBMCs(2x10/ml)wereculturedwitheachoftheHLAA*0201refoldingpeptides
ataconcentrationof10uMinRPMI1640mediumcontaining10%FCSand20U/ml
recombinanthumanIL-2(rhIL-2)in24-wellcultureplate.Halfofthemediumwas
changedatday4withsupplementationofrhIL-2at20U/ml.Atday7,cellswere
harvestedandtestedforthepresenceofpeptide-specificCD8+TcellsbyanIFN-γ
releaseELISPOTassay.
ZhouM,XuD,LiX,LiH,ShanM,TangJ,WangM,WangFS,ZhuX,
TaoHetal:Screeningandidentificationofsevereacuterespiratory
syndrome-associatedcoronavirus-specificCTLepitopes.JImmunol2006,
177(4):2138-2145.
2.PBMCswereseparatedfromheparinizedvenousbloodbyFicoll-Hypaquedensity
gradientcentrifugationfromHLAA*0201normaldonorsandHCCpatients.DCs
weregeneratedfromperipheralbloodmonocytesasdescribedbyRomani.Briefly,
PBMCswereseededintosix-wellcultureplatescontaining3mlRPMI-1640and10%
6o
FCSat5–10x10/well.Plateswereincubatedina37Cincubatorfor2h,thenthe
o
non-adherentcellswereremovedandtheadherentcellswereculturedat37Cin
RPMI-1640supplementedwith10%FCS,1000U/mlhumanrecombinantGM-CSF
and500U/mlhumanrecombinantIL-4.AllTcellstimulationwaswithday7DCs.
Asdescribedbelow,DCswerematuredwithlipopolysaccharide(LPS)onday6and
HCA587antigenwasaddedforeffectiveprocessingatthistime.
Day6DCswereresuspendedinRPMI-1640with1000U/mlGM-CSF,500U/ml
IL-4and10ng/mlLPSandincubatedat37oC.After24hcultivationtheDCswere
collected,washedandpulsedwith10
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