多發性骨髓瘤基因修飾瘤苗誘導體內抗腫瘤反應的實驗研究

[08-03-1015:19:00]

編輯：studa20

作者：任素萍，王立生，郭強，王華，賈向旭，徐娟，王恒湘，吳祖澤【摘要】

本研究目的是評價NOD/SCID小鼠皮下移植瘤模型對多發性骨髓瘤基因修飾瘤苗引發體內抗腫瘤反應的效果。首先給NOD/SCID小鼠腹腔注射人外周血淋巴細胞以在其體內重建人的免疫系統，然后皮下接種γ-射線滅活的基因修飾骨髓瘤細胞sko-007（表達綠色熒光蛋白或者p53、GM-CSF和B7-1基因），以PBS作為對照，最后植入活sko-007細胞進行攻擊。結果發現，與對照組相比接種感染腺病毒Ad-p53/GM-CSF/B7-1的sko-007細胞可以明顯抑制移植瘤生長，病理分析顯示移植瘤纖維組織增生伴彌漫性壞死增多，血管增生顯著。免疫組織化學染色顯示瘤灶內有人T淋巴細胞浸潤。結論：p53、GM-CSF和B7-1基因修飾的骨髓瘤細胞能夠誘導產生抗腫瘤免疫反應，有可能用于人類多發性骨髓瘤的免疫治療。【關鍵詞】

多發性骨髓瘤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(吳祖澤)，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