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《ki-67抗原肽致敏γδt細胞激發(fā)特異性ctl的體外實驗研究》由會員上傳分享，免費在線閱讀，更多相關(guān)內(nèi)容在學術(shù)論文-天天文庫。
1、Studyonhumanactivatedy6Tcellsinducepeptide—specificCTLresponsetoKi-67antigeninvitroAbstractObjectiveTodetecttheanti-tumoractivityofthey6TcellandtheimmuneresponseofspecificCTLinducedby78TcellsloadedwithKi-67antigenpeptideinvitro，thisstudycanprovideimportanttheoreticalbasis
2、forimmunothempyofcancer．Methods1．Peripheralbloodrnononuclearcells(PBMC)wereseparatedformtheHLA-A2positivehealthydonors．T6Tcellsweree沖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張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盆劌匡鱟墮塑±莖焦迨塞targetratio，oneffectortotargetratioof40
8、：l，thekillingrateoftheA375，MCF-7celllinewas42．8％．36．1％respectively；CFSE／PIdoublelabelingassaysho
