ki-67抗原肽致敏γδt细胞激发特异性ctl的体外实验研究

《ki-67抗原肽致敏γδt细胞激发特异性ctl的体外实验研究》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、Studyonhumanactivatedy6Tcellsinducepeptide—specificCTLresponsetoKi-67antigeninvitroAbstractObjectiveTodetecttheanti-tumoractivityofthey6TcellandtheimmuneresponseofspecificCTLinducedby78TcellsloadedwithKi-67antigenpeptideinvitro，thisstudycanprovideimportanttheoreticalbasis

2、forimmunothempyofcancer．Methods1．Peripheralbloodrnononuclearcells(PBMC)wereseparatedformtheHLA-A2positivehealthydonors．T6Tcellsweree冲andedinculturemediumwithIL-2pluszoledronicacid．WeassaytheratioofTSTcellsafterstimulationthroughflowcytometry(FCM)；anddetecttheanti-turmract

3、ivityof78TcellstohumanmalignantmelanomacelllineA375andhurmnbreastcancercelllineMCF-7invitrobymeansoftheLDHreleaseassayandCFSE／PIdouble—labeledassay．2．HighpurityofCD8+TcellsandactivatedySTcellswereobtainedthroughImmunormgneticbeadssortingtechniques．3．HumanKi-67antigenpepti

4、ded[280-288(LQGETQLLV)】andthecorrespondingMAP4modifiedpeptideswereproducedbysolid—phasepeptidesynthesis(SPPS)．Thepeptideswerepurifiedusingreverse-phasehigh-perforrmnceliquidchromatography(RP-m'LC)．ThepurifyofthepeptideswasconfirrmdbyarialyticalHPLC．Themoleculeweightwaside

5、ntifiedbyhigh-performanceelectrospmyionizationspectra(ESI-MS)．4．TheactivatedyfTcellspulsedwithKi-672s0_288antigenpeptideandcorrespondingMAP4modifiedpeptideswereco-culturedwithCD8+Tcells，unloadedy6TcellsascontroIgroup．WeselecthumancoloncancerceilsSW620(Ki．67+，HLA-A2+)，huh'

6、anbreastcancercellsMDA．MB．231张i-67+，HLA．A2‘)andhun-anrenalpmximaltubuleepithelialcellsHK-2伍i-67。，HLA-A2+)asthetargetcells，detectthecytotoxcityofdifferentgroupsofeffectorcellstothetargetcellsinvitrousingLDHreleaseassay；,theIFN-丫releaseofabovethreegroupsofeffectorcellswerem

7、easuredbyELISA．Results1．PBMCwerestimulatedbyIL-2andzoledronicacidfor10day,pr-oportionofT8Tcellswashighest,upto85．3％．2．LDHreleaseassayshowedthatkillingefficiencyofactivated78Tcellsgraduallyincreasedwithincreasingeffectorto5盆刿匡鲎堕塑±茎焦迨塞targetratio，oneffectortotargetratioof40

8、：l，thekillingrateoftheA375，MCF-7celllinewas42．8％．36．1％respectively；CFSE／PIdoublelabelingassaysho