多发性骨髓瘤基因修饰瘤苗诱导体内抗肿瘤反应的实验研究

多发性骨髓瘤基因修饰瘤苗诱导体内抗肿瘤反应的实验研究

[08-03-1015:19:00]

编辑：studa20

作者：任素萍，王立生，郭强，王华，贾向旭，徐娟，王恒湘，吴祖泽【摘要】

本研究目的是评价NOD/SCID小鼠皮下移植瘤模型对多发性骨髓瘤基因修饰瘤苗引发体内抗肿瘤反应的效果。首先给NOD/SCID小鼠腹腔注射人外周血淋巴细胞以在其体内重建人的免疫系统，然后皮下接种γ-射线灭活的基因修饰骨髓瘤细胞sko-007（表达绿色荧光蛋白或者p53、GM-CSF和B7-1基因），以PBS作为对照，最后植入活sko-007细胞进行攻击。结果发现，与对照组相比接种感染腺病毒Ad-p53/GM-CSF/B7-1的sko-007细胞可以明显抑制移植瘤生长，病理分析显示移植瘤纤维组织增生伴弥漫性坏死增多，血管增生显著。免疫组织化学染色显示瘤灶内有人T淋巴细胞浸润。结论：p53、GM-CSF和B7-1基因修饰的骨髓瘤细胞能够诱导产生抗肿瘤免疫反应，有可能用于人类多发性骨髓瘤的免疫治疗。【关键词】

多发性骨髓瘤Multiplemyeloma(MM)remainsanincurablemalignancydespiteadvancesinchemotherapy.Myeloablativechemotherapyfollowedbyallogeneicorautologoushematopoieticstemcelltransplantationhasincreasedtheincidenceofcompleteremission，butalmostallpatientsachievingcompleteremissionultimatelyexperiencerelapse［1-3］.

Becausethesechemotherapieshaveonlylimitedvalue，alternativestrategiesareneededtosolvetheseproblems.Immunotherapymayrepresentameansofmaintainingcompleteremission.

BasedonthefactthatmyelomacellscontainamultitudeoftumorantigensthatcaneffectivelystimulateantitumorTcells，severalinvestigatorshaverepor-tedimmunotherapeutic

approachesviainoculatingmye-ThisprojectwassupportedinpartbyChineseNationalBasicResearchandDevelopmentGrants’973’(No.2004CB518801andNo.2002CB713804)，ChineseHigh-TechProgram’863’(No.2003AA216050)andChineseNationalScienceFoundation(No.30400189).Correspondingauthor:WUChu-Tse(吴祖泽)，Academician

of

ChineseAcademyofSciences，ProfessorofDepartmentofExperimentalHematology，BeijingInstituteofRadiationMedicine.lomacelllysates，wholemyelomacellsorgeneticallymodifiedmyelomacellvaccinestoaugmenttheimmunogenicityofmyelomacells.Numerousapplicationsofgenesencodingtumorsuppressiveproteins，cytokinesandcostimulatorymoleculeshavebeenproposedincancer(includingMM)therapy［4-7］.

Althoughtheresultsofmostpreclinicalinvestigationscarriedoutinvitroandinmousemodelswereencouraging，theclinicalevaluationislesssatisfactory［8，9］.

Furtherstudiesfoundthatratherthantheabsenceoftumor-specificantigen，myelomacellsescapefromimmunesurveillancemainlybydown-regulatingtheexpressionofcostimulatorymoleculesandinhibitinginductionandmaturationofdendriticcells(DCs)［6，10-11］.

Henceacombinationofimmune-stimulatinggenesshouldbemoreefficientthananysinglegene.Inpreviousstudy，wehavede-monstratedthatwholemyelomacellvaccinationco-transferredwithhumanwild-typep53，GM-CSFandB7-1genesmediatedbyrecombinantadenovirusAd-p53/GM-CSF/B7-1inducesallologousandautologousspecificanti-tumorcytotoxicityinvitro［12］.

InthisstudyweinvestigatedwhetherthegenerationofAd-p53/GM-CSF/B7-1-mediatedimmunityisprotectiveagainstsubsequenttumorchallenge.NOD/SCIDmicearethemostimmunodeficientoftheSCIDvariants，andarethemostsupportivehostforhumanstemcells［13］.

MostSCIDmousemyelomamodelshaveusedmouseorhumanMMcelllines.In2studies，freshBMCfromMMpatientsurvivedinconventionalSCIDmice，andinonegroupitwasobservedthathumanMMcelllinescolonizedinhumanfetalboneimplantedsubcutaneouslyinSCIDmice［14-16］.

However，allthemicemyelomamodelscouldnotreflecttheinteractionbetweenhumanimmunesystemandhumanmyelomacells.

HuPBL-NOD/SCIDmousemodelhasbeenappliedinseveralothertumors［17-18］.

Inthisstudy，weintraperitoneallyinjected

humanPBLintoNOD/SCIDmice

toestablishhumanimmunesystem，innoculatedtheanimalswithgeneticallymodifiedhumanmyelomacellvaccine，andthenchallengedsubcutaneouslythemicewithliveparentalmyelomacells.Theresultshowedthatp53，GM-CSFandB7-1gene-modifiedmyelomacellvaccineproducesaninvivoantitumorimmuneresponse.

MaterialsandMethodsAnimals，myelomacellsandperipheralbloodlymphocytes(PBL)

InvivoexperimentswereperformedinfemaleNOD/SCIDmice(fromtheExperimentalAnimalCenter，ChineseAcademyofMedicalSciences，Beijing)，aged7to9weeks，whichwerebredandmaintainedinspecificpathogen-freeconditions.

Inthisstudy，ahumanmyelomacelllinesko-007，withhumanleukocyteantigen(HLA)A2positive，waskindlyprovidedbyProfessorBei-FenSHENfromBeijingInstituteofBasicMedicalSciencesandwasculturedinRPMI1640(Sigma)supplementedwith10%FBS，100U/mlpenicillin，and100μg/mlstreptomycin.

PBLsofnormaldonorsintheBeitaipingRoad2#HospitalofBeijing，China，appliedforestablishmentofhumanimmunesysteminNOD/SCIDmice，wereisolatedbyFicoll-Paqueseparationasdescribedpreviously.Sincesko-007cellsareHLA-A2positive，PBLswiththesameHLAantigenwereselectedandculturedinRPMI1640containing15%FBS，5%humanABsera，50U/mlIL-2，100U/mlpenicillin，and100μg/mlstreptomycin.Cellsweremaintainedinahumidifiedatmospherecontaining5%CO2at37℃.HLA-A2geneofPBLswasamplifiedbypolymerasechainreaction(PCR)asdescribedpreviously［19］.

Briefly，genomicDNAwasextractedfromPBLsofnormaldonorsaccordingtoWizardgenomicDNApurificationsystem(Promega，Madison，WI)，andHLA-A2PCRwasperformedinafinalvolumeof50μlwiththeuseof500ngDNA，0.1nmol/LMgCl2，0.01nmol/LdNTP，0.01nmol/Leachprimer，and1UTaqPolymerase(Promega)inPCRbuffer.Fivecycles，eachconsistingof60secondsat96℃，60secondsat66℃，and120secondsat72℃，followedby25cycles，eachconsistingof60secondsat96℃，60secondsat56℃，and120secondsat72℃，wereperformedinaMarstercyclerPersonalDNAThermocycler(Eppendorf，Hamburg，Germany).TheprimersforHLA-A2amplificationwere5’-CCTCGTCCCCAGGCTCT-3’(sense)and5’-TGGCCCCTGGTACCCGT-3’(antisense).β-actinwasusedasinternalstandard.Thereactionproductswereelectrophoreticallyseparatedthrougha1.5%agarosegelandstainedwithethidiumbromide.TheexpectedproductsgeneratedbyPCRwere813basepairs(bp)and396bpforHLA-A2and

β-actin，respecti-vely.RecombinantadenovirusandgenetransferAd-GFP(recombinantadenovirusexpressinggreenfluorescenceprotein)waskindlyprovidedbytheGeneTherapyUnit，BaxterHealthcareCorp.，USA.Ad-p53/GM-CSF/B7-1(recombinantadenoviruscoexpressinghumanwild-typep53，GM-CSFandB7-1proteins)wasconstructedbyourdepartmentviahomologousrecombinationinHEK293cells

(AdsE1-transformedhumanembryonalkidneycells).Theinsertedhumanwild-typeB7-1genewasdrivenbyaRoussarcomavirus(RSV)promoter，andp53andGM-CSFgenes，linkedbyinternalribosomeentrysite(IRES)，weredrivenbyacytomegalovirus(CMV)promo-ter［20］.

Thesetwokindsofrecombinantadenoviruswithhightiterandpuritywereobtainedbylarge-scaleamplificationinHEK293cellsandultra-centrifugationinCsCldensitygradientsolution.TheinfectiontitersofAd-GFPandAd-p53/GM-CSF/B7-1usedinthisstudywere1×1011pfu/mland5×1010pfu/mlrespectively.

Toproducethemyelomacellvaccine，sko-007cellswereinfectedwithAd-GFPorAd-p53/GM-CSF/B7-1atamultiplicityofinfection(MOI)of200for2hours.Culturemediumwasusedformockinfection.Afteranadditional48hoursincubationat37℃，5%CO2，transgenicexpressionofGFP，aswellasB7-1，GM-CSFandp53mediatedbyadenovirusweredeterminedbyflowcytometry，ELISAandWesternblot，respectively，aspreviousdescribed［12］.Establishment

ofhumanimmunesystem

andvaccineadministrationEighteenNOD/SCIDmicewereinjectedintraperitoneallywith(3-4)×107HLA-A2+PBLsin0.5mlPBS，pH7.4onday0andthenwererandomlydividedinto3groups:control，Ad-GFPandAd-p53/GM-CSF/B7-1group.Eachgroupof6huPBL-NOD/SCIDmicewasimmunizedtwicesubcutaneouslyontheabdomenwith1×106irradiatedAd-p53/GM-CSF/B7-1-orAd-GFP-infectedsko-007cellsin0.2mlPBSor0.2mlPBSondays7and14.Followingvaccination，allanimalsreceivedsubcutaneous

injectionof500UrecombinanthumanIL-2

permousefor3timesaweekuntilsacrificed.Tumor-challengestudiesOnday7afterthesecondinjection，varioustreatmentgroupsofmicewerechallengedsubcutaneouslyontheirbackswith5×106livesko-007cells.Tumorgrowthwasmonitored2to3timesaweekbymeasuring2maximumdiametersofthetumoratthesiteofchallengewithaverniercaliperandwasreportedasameanofthe2diameters.Micewereweighedusinganelectricscaleandsacrificedbyeyeballextirpationonday66.Thetumorswereexcisedandweighed.Thetumorvolumewasdeterminedbymeasuringthelength(a)，width(b)andthickness(c)usingaverniercaliperandcalculatedbytheformula:abcπ/6(mm2).Theweightindexwascalculatedastheweightratiooftumor/mouse.ProcessingofspecimensforhistopathologyWhenmicewerekilled，tumortissueswereremovedfromvarioustreatmentgroupsand

fixedin10%phosphate-bufferedformalinfor24to48hours，processedthroughgradedalcohols，andembeddedinparaffin.Serialsectionsoftumorswerecutatvariouslevelsandstainedwithhematoxylinandeosinforhistopathologicanalysis.AdditionalsectionswereusedforimmunohistochemicalstainingforhumanTlymphocytesusingmonoclonalrabbitanti-humanCD3antibody(Zhongshan，Beijing).Subsequently，tissueswereincubatedwithpolyclonalbiotin-conjugatedgoatanti-rabbitantibody(Zhongshan)followedbystreptavidin-horseradishperoxidase(Zhongshan).Thestainingreactionwasperformedfor10minwith3，3-diamino-benzidine-tetrahydrochloride(Zhongshan)inPBS(60mg/100ml).Finally，thetumortissueswerestainedwithhematoxylintodisplaythecaryons.DeterminationofhumanIglevelsLevelsofhumanIgGandIgAintheseraofhuPBL-NOD/SCIDmiceweredeterminedbyagardiffusionassay.Whenmiceweresacrificed，peripheralbloodwasharvestedbyeyeballextirpation.100

μlserawereaspiratedfollowingcentrifugationandmixedwith300

μldistilledwater.10

μlmixtureofeachsamplewa