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The_110

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The_110The,the

    The

,Sep.2O03,Vo1.12,No-现代妇产科进展2O039fll2鲞箜塑

    ;Theinvivost~dyofmyeloprotectionbyGST-Ttgenetransfectedhuman

    ;cordbloodhematopoieficstemcellstransplantation

    ;?

    ;英文稿?

    ;YangXingshengI,YuChenghao2,KongYawei3,JiangJieI,DengRniy~,CniBaoxiaI,WangLijieI,JiangSenI

    ;(1.DepartmentofObstericsandGynecology,2DepartmentofPharmacy,QiluHospital,ShandongUniversity,

    ;Ji~Jan250012;3.SchoolofLifeScience,ShandongUniversity)

    ;AbstractObjective:ToinvestigatetheinfluenceofGgenetransferintohumancord ;bloodhematopoiefiestemcellso11theirdngresistanceagainstantiUllllordrugsinvivo.Methods:

    ;GSr-7cgenetransfectionintohumancordbloodCD34cellswascarriedoutusil1garetrovirusvec- ;t0rPLJ.GSr-7cwiththeaidoffibronectin.Successfulgenetransferwasconfmnedbyinvitrocolony ;assayandRT-PCR.GSr-7cgenetramducedhumancordbloodCD34cellswerethene,c~tedinto ;4-week.oldtotalbodyirradiatedNOD/Scidmiceandearboplatinwasintraperitoneallyadministered ;sequentiallyat4weeksinto~4weeksafterengraftment.Results:Peripheralblood(PB)was

    ;significantlyhigherinGST-micethancontrolmiceafter2courseofcarboplatin.RetroviralGST- ;expressioninbonemwhematopoiefieprogenitorcellsofrecipientmicewasdetectedbyRT-PCR ;16weeksafterXenotransplantafion.Conclusion:Thetransfectionof(r.germcouldconfer.to ;someextent,resistancetocordbloodstemceilsagainstcarbeplatininrive. ;KeywordsJFetalblood;Hematopoiefiestemcells;Genes,&ST-7c;Dmgresistance ;CIhber:R322.2Dl0c咖咖tcode:AArticleID:10047379(21XB)05039803

    :w(BM)orperipheralbloodstemcell ;Bone?m

    ;transplantationshortenedtheperiodofehemotbempy-in

    ;ducedneutropenlabutd/dnotdecreasethedeepthofthe

    ;neutrophilnadir,Ies0lviIonlypar?ythemyelosuppres

    ;sionproblems.Itisoftendifficulttoveadditional

    ;chemotherapyafterBMtransplantationowingtosterncell

    ;exhaustion.

    ;Ithasbeenreportedthatautologoustransplantation

    ;ofBMcellstransfectedwithdmgresistancegeneshas

    ;beenPror~edtoove~y3lnetheseveremyelosuppression

    ;inevitablyassociatedwithsequentialhishdosecheanother-

    ;apy(tmC).Ggene,theabundantmemberofGST

    ;(ghtathioneS-transfera~)family,whichneutmlizeselec?

    ;trophilictoximbyghtathioneconjugationorbin~S,has

    ;beenprovedtoberel~tedtodrugresistancen~instplat

    ;inum,alagagentsandadriamycin.Ithasbeen

    ;demonstratedthatGST-Ttgenetra~qductionintohumanpe? ;ripheralbloodCD34ceilsprotectedthemagainstadri

    ;amycinand4-hydroperoxycyelophosphamideinvitro.Fur- ;therstudyshowedthatandGST-~germtransfectedbone ;nwceU(BMC)transplantationcouldprotecttheBMCs ;fromthetoxicityofmuhicyeliesequentialhish.-dosecy.. ;clhosphamideinaroutinesyngeneieboneIllalTOWtrans. ;plantationmodel[?.

    ;Cad~plntin[eis-diammine(cyelobutane.1,1.dicar. ;398

    ;boxylato)platinum(?)]isasecondgenerationplatinum

    ;complex,whichisulllikethefirstgenerationplatinum ;drugs,suchaseisplat~n,haslessnephwtoxieitybuthas ;substantial,adversemyelosuppressivee,ffectL2J. ;Humancordbloodhasemer~tasapotentials0l[?e

    ;ofhematopoietiestemcoilsforpatientswhoaI?inneedof

    ;hematopoietiestemcelltransplant.Humancordblood ;transplantationoffersagoodalternativetobone//la/low ;transplantation[3I.

    ;Inthepresentstudy,wehaveattemptedtoevaluate ;theehemoprotecfionofBMCsfromsequentialcarboplatin ;invivobyGcgenetmnsfectedhumancordblood

    ;CD34celltransplantation.

    ;1MaterialandMetlmds

    ;1.1Tmnsfectionofthe7cgoneintocellsCollection ;ofkcordbloodandCI)34Upurificationand7cgerm ;trar~irintoCI)34cellswa8carriedoutreVortcdvi0tls.

    ;1.2TransplantafionofCD3-4cellsintoND)/SamiceXeno-

    ;transplantationofthe7corneoRgene-~CD34cells ;wa8performedwithamodificationofthemethoddescribedprevious. ;1y….Briy,cellsectedt}gIIthetailveininto4.we

    ;ddNOD/SCIDmiceirradiatedwith2.4Gyoftotalbodyill~a- ;tion.,I1lerecipientmiceifectedintraperitor~ywith400~aof ;PBScentaining20ofanti-asialoGMIaJl(wako,Osaka, ;Jlp8n)iIl1medialybeforethecelltransphnmt/on.Themice ;administeredintraperitoneallywith1.2nag铡出血[.pe.

    ;

    ;现代妇产科进展2OO39月第12卷第5,Sep.2OO3,Vo1.12,No.5 ;fiphe~~ood(PB)werecollectedforWBCccRl】吨oliday0,4,7

    ;and14~mluistrafionofcIb0I.

    ;I.3I)e~orlofG7cexpressioninmicebonemalTowcells

    ;MiceweIelIldeeDanesthesiabydiethyl-ethcr16weeks ;afterthea】隘IDlan0n.F臼蛐andtibiaewerecollectedandaspi. ;ratedwith5%no0IltIIMDMt0liberateboneIllfIl’owce1ls.

;Cell嗣玛pweIefiltered吐吒llasterile40mncellraiIlerto

    ;getrid0fdl8anddebrisandthenooe88edforRT-PCR

    ;aI8.TotalRNAextractedfromindividualcolonieswaftusedfor ;RT-PCRamay.ThenRT-PCRwaftcarriedoutinareactionsys. ;temw/tlowedby3-stepafterinitialPCRactiva. ;fionstepat95oCfor15rain.ThecyclingcoadifiomweIe:&natura-

    ;tion94?for45s,舳溉at55oCfor45sandex~onat72?

    ;forImin.andfinale~siorlat72?f.0r10minaRer35cycles.The ;100

    ;8O

    ;06O

    ;4O

    ;?20

    ;&0

    ;a

    ;strboC

    ;p

    ;Olot~

    ;a

    ;rscof

    ;.

    ;ItT-t~productsweIedI,cIr0l0l11%ag6eand

    ;stairledwitlletlli&umbnln.

    ;1.4StaticalaI|Forthestati~calcomp~_dsoninPBM- ;NCs.Student’8t-testapplied.Theeililicamlevdset1o

    ;0.o5.

    ;2Results

    ;2.1SerialchaI1geinthephbloodWBCinre.

    ;cipientmiceFiveofmiceweretransplantedwitIlC7c ;genetransducedhumanCD34celltIllmtailvein, ;controlledwitIlmicetransplantedwitIlneoRgenetrans. ;ducedCD34cells.AtIlemiceweretreatedwitIlcaIb0. ;platin(1.2rag/mouse)intraperitonealyonday28after ;transplantation.Peripheralblood(PB)WBCwerechecked ;onday0,4,7andday14ofcarbeplatinadministration. ;Afterfirstooul’$~ofcar~platin,therewasnosignificant

    ;differenceinPBWBCbetween2groups,huttherewas

    ;significantdifferenceaftertIlesecondeour8~ofcarl~ ;platin.(.1).

    ;0471428323542566O637O

    ;daysafterlstcourseofcarboplatin.,,

    ;Fig.1SerialchangeinWBCaftertmammmtllpla.

    ;?

    ;miceunderwentIl0III!allonwith(;ST-7cle砸眈如ctedhl衄锄

;coldbloodCD34cell;umicellIldeIxenotransplantationwithneoRgeaetransfeetedh

    nc0Idb1o0dED34+cells

    ;2.2GST-~geneexpressioninthebonenlalTOWceilsinalltheGST-~rmice,whileneoRmicedidnotexhi

    bitany

    ;recipientmiceGST=~mRNAwasdetectedintheBidsofGST-~n~NA(fig.2).

    ;A

    ;M12345

    ;~364bp

    ;M12345

    ;7l5bp

    ;364bp

    ;2RT-PQt

    ;.

    ;?0f7cmRNAinPBMNCsfromrecipientmice2weksafterthe3rdeomseofcarboplatin.

    ;A,n~’ansfeeted;B,

    ;bl7qtransleeted

    ;3Da

    ;Theefficacyofmanychemotherapeuticagentsis ;limitedbytheirtoxicitytononTlaltissues ;,

    ;resultingin

    ;nalTOWtherapeuticindex.Myelosuppressionisthern0st ;frequentacutedoee-limitingsideeffectresponsibleforde. ;1aysinchemotherapyadministrationord0sealH_eai0ll

    ;withdecreasedantitumourresponse.na血吼.c0liniI1g

    ;regiarethecommonlyusedchemotherapeuticregi. ;399

    ;

    ;现代妇产科进展2OO39月第l2鲞筮塑.Sep.20O3,Vo1.12,No.5 ;ITI~/I$forovariancsIlcer.Dnlgresistancetoplatinumand ;alk)ragentsarel~]8tedtoGST-xgeneexpression? ;GSr-7gemetransductionmayconferresistanceto ;bematopoieticprogenitors~instthesereagentsandpro

    ;tectionofmyelosuppressioninducedbyPCorPAC ;chemotherapy.Invitroexperimenthadbeence~mnedthat ;GSIIletnmsductionintohumanCD34cellscould ;conferdmgresistancetondrhmycinand4HC(active

    ;formofcyclophoophamide).Carboplatlnisasecondgen

    ;erationplatinumcomplex.Clinically,carhophtlncansub

    ;stitutecisplatininthetreatmentofovariancaIlcer,non

    ;smallcellande~1.sivestageaUcelllungcancer.Un

    ;likethetgenerationplatinumdm,suchascisplatin, ;carhoplafinhaslessnephrotoxicityandototoxicitybuthas ;substantial,adversemyelosuppressiveeffect.Ithasbeen

    ;reportedthatcarboplatlnrequireda10timeshigherdrug ;concentrationthancisp!afintoinduceasimilardegreeof ;growthinhibitiononleukemiccells,however,the ;hematopoieticprogenitorsrespondede~mllytocisphtin ;andcarboplatinatthes!咀drugconcentration.Thisresult

    ;isconsistentwithapreviousreportwhichshowedthatcar

    ;bopt~was10timeslesspotentthancisphtiningrowth ;inhibitionofovariancarcinomaxonogr~tsinimmune-sup

    ;pressedmice.Fromtheseresultswecance~h&thatthe ;cencentrationofcarboplatlnshouldbemuchhighterthan ;thatofcisphtintogetthesneresultf0lrtl1Ietreatmentof

    ;ovariancarcinoma,andthemyelosuppressiveeffectisalso ;higherthanthatofcisp!a6naccerdingly.

    ;Hemmopoieticstemcellsarcknowntobeoptimal ;targetsforgeaetherapiesbecausetransducedgeneexpres

    ;sionmaybesustainedI?|0longterminvivodueto

    ;theirabilitytoreconstitutetheheamtopoieticandimmtlne ;systemspermanently.Successfulgonetrs/isferintothe ;mostprimitivehematopoieticcellsorlong-term~opopuht

    ;msstenlcellsmayleadtoalifelongcuIeforavarietyof ;diseasesmanifestedintheprogenyoftheseceils.CD34 ;cellshavebeenusedasanindexforhematopoieticstem ;cells.andallogeneicCD34+celltransplantationisactual- ;lybciIlgperfomaedinclinicalsett~.

    ;UedaetalE6]demonstratedthatcombinationofSCF ;,

    ;FL,TPO,IL-6andIL-6Rinducedsignificantexvivoex- ;paIlsi?ofhmmnhematopoieticstemcellscapableofre-

    ;pop~inginNOD/SCIDmice,suggestingthatthiscem

    ;binationofcytokinesdirecdyactonhumanhematopoietic ;stemcells,eventhoughtheymayaffectdifferentstagesof ;hematopoieticstemcellsintheirexpansion.Inthepresent ;study,wefoundthatCD34cellsincubatedwithaeombi

    ;nationofthesefactorsfor48hourssuccessfullystimulate ;humanhematopoieticstemcellsexpanseandenhanced ;GSr-7genetransfectionintohumancordbloodCD34 ;ceils.

    ;ThemechanismofdrugresistanceduetoC7cis

    ;basedonitsability:(i)tocatalyzetheconjugaionof ;electrophilictoxinswithglutatlli0ne,r~ultmSinnllol~po- ;hrandreadilyexcretedmetabolites,(ii)tosequester ;toxinsthmu#hishaffufitybinding,and(iii)toremove ;toxicperoxidestll删曲intrinsic0IIrIicpemxidaseactivi

    ;.Ourinvivodabindlca~!tlIGST-~geaetrs/lsferre

;ducedcarboplatininducedmDnali,s;LI;e{|til1gtlInddi

    ;tionalchemotherapymaybe西vduringtheposttransla

    ;tionptod.

    ;GST-=genetrs/isfermi#tbeusefulinestablishing ;H#-d~chemotherapy-resistantnmnuwcells,whichcan ;facilitatemulticyclicsequentialHDC.Butunforumately, ;theanticancerdmtowhichGST-~genecangabout ;refractorinessislimitedtoadriamycin,cisplatinum,etopo- ;side,andngagentssuchascyclophoophamideand ;IllpkIkm.Inclinicalsettil1,multipledugcembination ;re.e/isareoftenusedforstandardandhi#dose

    ;chemotherapies,andtheseIen朔【1soccasionallycontain

    ;血垮thathavembeariIlg?resistancerelevamtocsI7.

    ;1h,itisnecessarytoccmbimanotherresistance ;genesuchasMDR1andDHFBintogeneratedrure

    ;sistal~boneminhumancaIpmdlngse

    ;quential珊幽pledrugcombinationdllod1.

    ;[1]?岣删T,Sal,~ldS,lT,eta1.Gsr.,cgemtra-

    ;ducedhematopoieticprogenitorcell:哪印lamovm

    ;thebonemarIDwtoxicity0f~ydoph~phmid~inmice[J.

    ;HumGemTher,2000,11:1671-1681

    ;[2]SuWC,QngSL,CheaTY,eta1.c’pBri8ofinvitro

    ;growth-inhibitoryactivity0fca~oplmlnandcisp)~_n?

    ;leukemiceelhandk血越0peti?ptogemlmm:tl~m

    ;pressiveactivity0fc日曲0llamaybeeerthanits811-

    ;fileukemieect[J.】两JOmd,2OOO,30:562-567

    ;[3]YuLc,WDA,Slllwt]_~-E,a1.Umdatedcordbloodtra.

    ;planta甲咀ialoebythepediacbloodandm缸嗍缸】m

    ;eomortium[J].PediatrHmntdOned,2001.18(4):235.245 ;]YuC,Yah8x,CulB,eta1.Invitrostudy0fGSr.,cgem ;ti’a~erollre醯吐aI.oe0fb,m~neonicells

    ;[J].ProsObstetGynecol,2OO3,12:240

    ;[53StefaniehEG,Cadson-Zenne~CC,McEvoyK,eta1.Dose ;scheduleofrccombin~murin~thro~poietin0ftomyelo-

    ;suppressiveand舶吐tbinmlce[J].Cancer

    ;ClaemotherPharmaed,01,47:777

    ;[6]UedaT,TsujiK,YoshinoH,a1.dh,m~NOD/

    ;SClD-repopuht~cellsbystemoellfactor,Flk2/Flt31ir_d, ;~ietln,il-6,andsolubleil-6r?[J].’nJQi|I

    ;Invest.20o0,1O5:1013-1021

    ;

    ;

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