【doc】淋巴因子激活的杀伤细胞对有丝分裂期细胞的杀伤作用

【doc】淋巴因子激活的杀伤细胞对有丝分裂期细胞的杀伤作用 淋巴因子激活的杀伤细胞对有丝分裂期细 胞的杀伤作用 第32卷第3期 1998年6月 哈尔滨医科大学. JO~ALOFHARBINMEDICALUNIvERSITY Vd.32,No.3 Jun.1998175 EffectofHumanLymphokine-Activated KillerCellsonMitoticPhaseTumorCell L/m李殿俊ZhangXiaowei张晓伟LiuGuorong刘国蒙 (CancerInstitute) AbstractInordertoimproveLAK,IL-2efficiencyintumorimmunotherapy.thecytotoxicityofhu- maIlLAKcellsagainstmitoticpha8eofhumanlungadenocareinomasAnip973cellswasstudiedinvitro. Anip973cellswe? synchronizedbyspacificmitoticphaseantitumordrugVincristin(VCR).Thediffer- enceinmorphologybetweenmitotictumorcellsandunmitotictu/norceilswereobservedbytightmicros- copiesandtransmissioneloctromicmseopies.CytotoxicityofhumanLAKcellsagainstmitoticphase Anip973cellswassignificandyincreaseinstandard4-hou:Crreleaseassay.Inthefltl~efmorphological investigationbyscanningandtransmissianelectromicroscopics,theresultsindicatedthatIAKcellswel'e inclosecontactwithtargetcellsintheformofinterdigitateofmicrovilliandprotrusionsstruct ux~.The formofde砒hofmitoticphasetumorcellswaslyticnecrosisandapoptosis KeyWordsLymphokine;Killercells;Mitotictumorcell;Necrosis;Apoptsis AdoptiveimmunothempyusingLAKcellsandrlL-2hasrecentlygainedmuchattentiontotheob— servedantitumoractivityin8omeadvancedcancerpatients.Ahrgaammmtresearchworkhasbeendone incellularandm0locufartoimpmvetheantitumoractivltyofLAKcells[.Butthlsantitumoractivityw船 alsoinfluencedbythebiologicalcharacteristicoftu/norcells.Inthispaperwestudiedthecytotoxicityof humanLAKcellsagainstmitoticphasetnnlorcellsaccordingtothetunlorcellcycleandunitedanfitomor chem[caldrugtherapy. 1Tunlorcells:HumanlungadenocarcinomasArfip973cellsmsintainedbyserialinvitropas— sageinstandardculturemediumRPMI1640. 2VCR:VincristinproducedbyGuangzhouMingXingPharmacyFactory. 3rlL.2:WaskindlyprovidedbybiochemistryinstituteAcademiaSinicaShahghai, 4LAKcellpreparation:PeripheralbloodmononuclearcelIs(PBMC)wereisolatedfromhepa— rlnizedbloodofhealthvolunteersandseparatedoilFicoll-Hypaquedensitygradient1x106perm1.PBMC wereincubatedwithrIL一 2atllXlOpgpermIinRPMII640containing10percentheat-inactivetedhuman ABserulnfor5to7daysat37? in5%C02atmosphere.cLAKccllswerethenharvestedandwashed twicewit}IPBSandresuspendcd;ncompletemediumforthecytotoxieity嘴ay. 5Mitoticphasetlmlorcellpreparation:ExponentiallygrowingculturesoflineAnip973was addedattlnalconcenlration0.7gg/mlVCR,after20hcultureat37? 5%CO2,discardthecultured mediumslowly,andinsteadanewone,vacillateflaskBOWtotheleftnowtotheright,lettheliquidwash 176JOURNAL0FHARBINMED1CALUNIVERSVo1.32 outthemimdetuBlorcellswhichbecomeround,centrifugeandcollectedthecells.econtrolgroup ceilswereharvestedbytrypsindigested. 6Preparationofchromosomesampleoftiilllorcells:Chromosomesamplesweremadeasde一 ?)edelsewhere[. 7CytotoxidtyofLAKceilsagamstmitotictlllnorcells:LKactivitywasmeasuredills4hsCr reles~eassay嬲described.hbrief,targetcoils2x1扩 wereincubatedin10(1RPMI1640containing 10%FcS.Atotalof10ogCiofNCro4wastheuaddedandincubatedforanhour.Cellswerewashed threetimesinmediumandadjustedtoaconcentrationof1× 1suspensionandthenaddedperwellin triplicatetoroundbottom96-wdlmiemtite~plates.Atotalof100"l0fLAKcellsinappmprinteeol'l~~o— tratlozlaccordingtoE:Tratiowasthenaddedandfurtherincubatedfor4hours.T0assay'Crrelease, atotalof10o"1ofthesupernatantwascollectedfromeachwellandcountedinagammacounter(Pack— ardInstruments)..I11epercentageofspecific$~CrreleaseW;EI~determinedas: specificrelease=×1O0% 8Semmingandn.a岫嗣帕缸dectronmk~y:Forelectronmicroscopy,cellsuspension ofmitotic~'ulnorcellsandcontrolgrouptumorcellsweremixedwithLAKcellsatallE:Tratioof10:1 andincubatedfordifferenttimes.Thecellswerefixedinsituwith3%glutaraldehydeandthenasrountin waytomakescanningandtransmissionelectronmicroscopysampleforexaminationonH一 600(Hitachi Company). RESULTS 1A锄Ionclulllorcellswel'l~inducedbyVCR First,weobservedthebesttimeandeoncent,~ltionofVCRinducedtumorceils,exponentiallygrow— ingcuhurecellsoflineAnip973wereaddedatdifferentfinalconcen~ationVCRandcultureddifferent time,harvestedtheceilsandmadechromosomesample,countedgrowthfractioninlightmicroscopy.As deacribedintable1.thebestconditionofVCRindueedmitotictUl~aorceilswas0.7~g/mlinfinalcon— centrafianfor20h,growthfractionwas35.7%,thecontrolw舾2%一4%. Table1Grm~ahacll?Anlp973cdlsInducedbyVCR ^由3zells~ul/,ul~withVCRwe=etil~och坷呻Bo8'呷 lemadeeuntcdtheix=omta~0frtcpIImecall 2MorphologicalchangesbyVCROiltmnorcells Thetwodifferenttypesofmitoticcellandunmitotietunle~cellcanbeeasilydistinguishedmorpho— l咄i?u#tandemissionmicroscopy. Fig1wasthegrowingcellsinlightmicroscopy,leftwasthenormalgremngtumorcells(eont~l No.3 Di1111,ctalEFFECTOFHUMANLYMPH0砌NE-ACT ONMrroTIcPHASETUMORCELL ATEDK1LLEBCELLS 177 p),thecellsweIegrowinginanchoredstage(adhe~'etothecultureflask),growinginirg山 rp0ly— poleform.Themitoticcellsweremum,molereflectlight,thelink0fcellsweIebrokenandthecells wereveryeasilywashed—outbyrocking.. Thedifferenceinm0rph0l0 舒,betweenmitotictumorceilsandunmitotictumorcellswasobservedby transmissionelectronmicroscopies.Thecoodensadonoftllechromatinanddisappearanceofnuclear membranewereseeninmitoticcells.thecontrolgroupcellsnuclearmembranewaBcomplete,kernelwas easyto8ee.cbromatinwasdispersed. 3cyt0to~dtyofhumanLAkcellaga~tmitotict[1ll~orcell Cytotoxicitytomitoticttu~ortargetwasme~umd? previdllslydescribed.Theresuhsareexpressed intale2?themeanpercentage0flyshoftripletexperiments. Table2q咖dlyofhumanLAKcellssgsLastmitoticPhaseof~p973cells ^p973+vcR:Anip973悭? eultarewithVCBatthefindconcen~tlon0-7gg/rdfc~20hbefore8c.tedtoIbyLAK*disin4h Crretrainassay CytotoxicityofhumanLAKcellsagainstmitoticphaseAnip973ceilswassomuchhigherthanthatof unmitotictumorinstandard4h5lCrreleaseassay(P<0.o1ftest)andthepercemtageofcytotoxicityof LAKcellswassignificanflyincreasedwiththeincreaseofgrowthffacdonofthetlnuorceUs. 4Morphological曲mgehlducedbyLAKcellsonmitotictargetce? Wi凼 2hofco.cultu~.thecontactbetweenmitotictumortargetcellsandLAKcellswereseeninthe formofintcrdigitateofmicmvilliandprotrusionsatthesurfaceofcontactKl'ea(Fig.2,3).Itwa spreanm— ablethatthecoIl=lact8andiunctionsb~ecnLAKandtargetcellscouldmakethemboundtogetherfu-mly, andtheyweIethepathwayforexchangingmessagesbetweeneachother.About4hours,thetargetceils becamedead,thetwod~cmnttypesoftargetceilsdeath,necrosisanda~ptosiscouldbeeasilydistin— guishedinemissionelectronmicroscopies.Incellsundergoingnecrosis,therewasdissolutionoforga- dizedcytoplasmics|l"uctttreandlossofmgnjzahleorgandieswhilethenucleusr~in8intact(Fig.5). Incontrastcellsundergoingprogrammedcellsdeathshowedtheformationofresides,whichgavettchar. acteristic"bubbhng"appearanceandcondensationandsegmentationofthenucleuswithformationofthe so_called"apoptotlcbodies"(Fig.4).Theseresultsweresanlc? thatofcontrolgroupttullorcells. DIsCUssION Inourstudies,humanade~ocarcinomssAnip973cellsweresynchmnisedbyspecificmitoticphase antitumordrugVincristin(VCR).Becauseitcouldinhibitemicmtttheproreinaccumulatingandmadethe cellcyclestayatmitoticphase,thisconce~alrationofVCRwssll'tcytotoxicitytotuluorceU.Th/sfunction ofVCRwaBveIsible.Then皿0rceIIcouldcontinuegrowingfleeofVCR 啪.Thecytctoxicmcchsnls1]lS 178JOURNALOFHARBINMEDICALUNIVERSITY,.32 .fLAKcellshavebeenthesubjectofintenseresearchSofar,threedifierenttypesofmoleculeshave beenidentified[,.Perforinhasbeenpurifiedandshowntoformporesincellmembranesdisrupting theosmoficbalanceofthecellsandsubsequentlyburs6ngit[,.Afamilyofserineesterasehas been done.butaspurifiedserineesteraseswereincapableofkillingtheirrolewasstillnotunderstood.Fi— hally.differentmoleculesantigenicallyrelatedtoTNFandlymphomxlnhavebeenidentified.eNe moleculeswereknowntobecytotoxictospecificcellsandcausedDNAfragmentation血 veryslowki— netics.Itispmbablethatmuir:ipiecytolyticmediatorsmaybeemployedbyLAKcells,becauserioisolat— edmediatorcanaccountforallthecharacteristicsofLAKcellsmediatedkillingCytotoxicityofhtmaan LAKcellsagainstmitotictLRllOrcellsisincreased.Itisprobablethatthemorphologicalchangesofmitotic ti.R12orcellsmaketheLAKcellseasycontactthetlRllorcellsandthemitotictumorceilsissensitiyeto multiplecytolyticmediatorsarethemainteason.Thisresultisveryimportantinadoptiveimmunotherapy. Itcanprovideanev~wayinusingLAKeelltocureadvantedtt.113~orpatient. (Fig.1,5seeinsidebackcover) REFERENCES 1ChaoXuetao.eignMedicineIrrmtmol~yBranch,1994};4:176 2ZhengE."13ssueC~lmr~Techndc~a'peoplebygi~icpubtkshbltreatl(Secondedition),1986 3uu(amh~gHtmamdm螂neme小.dd0HarbinMedicalUniversity(fom~hedition),1989:5 4Amainzyc】 etat.Induction.fapoptoslsandnecmsmkille~lyrnphoeytesT.1mmunol,1991;146(1):393 5WangZhenyuan/vialignamtumorchemicaltherapyTmnc~~piudHeao~jmngPrncer1985 6r^mPA.Mechoni~n.flymphocyte-mediatedeyto~cacicityuRevhnmunol,1985.3l31 7Pod~~k.ERThemolemal~mechanism.flymphocyte-mediatedtt.~lta~celllysis.b?.Today1985:6:21 8youngJDE.CohnZACelluI~andhumc~tmechanismao壬cc时:mm吐町andfuaeti~~lanalngiesAdvI,rmam~,1987:41: 269 9TschoppJ.Nabho~M.Perfiarln-medlatedtarg~-'tcellIj~is?_七TI,砷}峨s.AnnuRev丘础?,l990;8f279 1ORussell】HInternaldlrdntegraticamodelo壬c蛐clymphocyte-inducedtarg~-td 曲.hmunatR,1983;72:97 1lJerI了】eDE.TschoppJGl?I栅5afamilyofsednepzot~srde~edh锄granulesofcymlY~icTLymphocy~eauporITee1]recePr rmhm舯ndRev,1988;103:53 1]一11'…", 淋巴因子激活的杀伤细胞对有丝分裂期细胞的杀伤作用 李殿俊等I之]一' 摘要为提高淋巴因子激活的杀伤~(LAK细胞)临床应用的疗效,采用抗肿瘤化疗药物长 春新碱诱导人肺腺癌细胞Anip973进入有丝分裂期,研究LAK细胞对有丝分裂期肿瘤细胞的杀伤 作用.结果表明,肿瘤细胞进入有丝分裂期后,形态上发生明显的变化,I.AK细胞对有丝分裂期肿 瘤细胞的杀伤作用明显增高.电镜下,I.AK细胞与有丝分裂期肿瘤细胞以绒毛相接触,呈是牙交 错状.靶细胞死亡形式是溶解坏死和凋亡. 关键词坠璺;苎望竺竺;互竺坌塾塑壁垄垫;溶解坏死;凋亡