The
,Sep.2O03,Vo1.12,No-现代妇产科进展2O03午9一fl箜l2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)
;【Abstract】Objective:ToinvestigatetheinfluenceofGgenetransferintohumancord ;bloodhematopoiefiestemcellso11theirdn】gresistanceagainstanti—Ullllordrugsinvivo.Methods:
;GSr-7cgenetransfectionintohumancordbloodCD34cellswascarriedoutusil1garetrovirusvec- ;t0rPLJ.GSr-7cwiththeaidoffibronectin.Successfulgenetransferwasconfmnedbyinvitrocolony ;assayandRT-PCR.GSr-7cgenetramducedhumancordbloodCD34cellswerethene,c~tedinto ;4-week.oldtotalbodyirradiatedNOD/Scidmiceandearboplatinwasintraperitoneallyadministered ;sequentiallyat4weeksinto~4weeksafterengraftment.Results:Peripheralblood(PB)贼was
;significantlyhigherinGST-micethancontrolmiceafter2courseofcarboplatin.RetroviralGST- ;expressioninbonem蛐whematopoiefieprogenitorcellsofrecipientmicewasdetectedbyRT-PCR ;16weeksafterXenotransplantafion.Conclusion:Thetransfectionof(r.germcouldconfer.to ;someextent,resistancetocordbloodstemceilsagainstcarbeplatininrive. ;【KeywordsJFetalblood;Hematopoiefiestemcells;Genes,&ST-7c;Dmgresistance ;CIh皿ber:R322.2Dl0c咖咖tcode:AArticleID:1004—7379(21XB)05—0398—03
:w(BM)orperipheralbloodstemcell ;Bone?m玎
;transplantationshortenedtheperiodofehemotbempy-in—
;ducedneutropenlabutd/dnotdecreasethedeepthofthe
;neutrophilnadir,Ies0lviIonlypar?ythemyelosuppres—
;sionproblems.Itisoftendifficulttoveadditional
;chemotherapyafterBMtransplantationowingtosterncell
;exhaustion.
;Ithasbeenreportedthatautologoustransplantation
;ofBMcellstransfectedwithdmg—resistancegeneshas
;beenPror~edtoove~y3lnetheseveremyelosuppression
;inevitablyassociatedwithsequentialhishdosecheanother-
;apy(tmC).Ggene,theabundantmemberofGST
;(ghtathioneS-transfera~)family,whichneutmlizeselec?
;trophilictoximbyghtathioneconjugationorbin~S,has
;beenprovedtoberel~tedtodrugresistancen~instplat—
;inum,ala勘gagentsandadriamycin.Ithasbeen
;demonstratedthatGST-Ttgenetra~qductionintohumanpe? ;ripheralbloodCD34ceilsprotectedthemagainstadri—
;amycinand4-hydroperoxycyelophosphamideinvitro.Fur- ;therstudyshowedthatandGST-~germtransfectedbone ;n哪wceU(BMC)transplantationcouldprotecttheBMCs ;fromthetoxicityofmuhicyeliesequentialhish.-dosecy.. ;cl叩hosphamideinaroutinesyngeneieboneIllalTOWtrans. ;plantationmodel[?.
;Cad~plntin[eis-diammine(cyelobutane.1,1.dicar. ;398
;boxylato)platinum(?)]isasecondgenerationplatinum
;complex,whichisulllikethefirstgenerationplatinum ;drugs,suchaseisplat~n,haslessnephwtoxieitybuthas ;substantial,adversemyelosuppressivee,ffectL2J. ;Humancordbloodhasemer~tasapotentials0l叫[?e
;ofhematopoietiestemcoilsforpatientswhoaI?inneedof
;hematopoietiestemcelltransplant.Humancordblood ;transplantationoffersagoodalternativetobone//la/low ;transplantation[3I.
;Inthepresentstudy,wehaveattemptedtoevaluate ;theehemoprotecfionofBMCsfromsequentialcarboplatin ;invivobyG丁cgenetmnsfectedhumancordblood
;CD34celltransplantation.
;1MaterialandMetlmds
;1.1Tmnsfectionofthe7cgoneintocellsCollection ;ofk咖cordbloodandCI)34Upurificationand7cgerm ;trar~irintoCI)34cellswa8carriedout硝reVortcd肿vi0tls引.
;1.2TransplantafionofCD3-4cellsintoND】)/SamiceXeno-
;transplantationofthe7corneoRgene-~CD34cells ;wa8performedwithamodificationofthemethoddescribedprevious. ;1y….Bri棚y,cells呲ectedt}咖gIIthetailveininto4.we出
;ddNOD/SCIDmiceirradiatedwith2.4Gyoftotalbodyill~a- ;tion.,I1lerecipientmice眦ifectedintraperitor~ywith400~aof ;PBScentaining20ofanti-asialoGMIaJl(wako,Osaka, ;J习lp8n)iIl1medialybeforethecelltransphnmt/on.Themice ;administeredintraperitoneallywith1.2nag铡出血[.pe.
;
;现代妇产科进展2OO3年9月第12卷第5期,Sep.2OO3,Vo1.12,No.5 ;fiphe~~ood(PB)werecollectedforWBCccRl】吨oliday0,4,7
;and14疵~mluistrafionofc吕Ib0I岫.
;I.3I)e~orlofG7c孵expressioninmicebonemalTowcells
;MiceweIel口Il】deeDanesthesiabydiethyl-ethcr16weeks ;afterthe缸a】隘IDlan0n.F臼蛐andtibiaewerecollectedandaspi. ;ratedwith5%no0IltIIMDMt0liberateboneIllfIl’owce1ls.
;Cell嗣玛p既weIefiltered吐吒ll小asterile40mncell砒raiIlerto
;getrid0fdl唧8anddebrisand慨thenooe88edforRT-PCR
;aI姆8.TotalRNAextractedfromindividualcolonieswaftusedfor ;RT-PCRamay.ThenRT-PCRwaftcarriedoutinareactionsys. ;temw/tlowedby3-stepafterinitialPCRactiva. ;fionstepat95oCfor15rain.ThecyclingcoadifiomweIe:&natura-
;tion越94?for45s,舳溉at55oCfor45sandex~onat72?
;forImin.andfinale~siorlat72?f.0r10minaRer35cycles.The ;譬100
;星8O
;06O
;苫4O
;?20
;&0
;a
;strboC
;p
;Olot~
;a
;r血scof
;.
;ItT-t~productsweIedI,cIr0l0l11%ag哪6eand
;stairledwitlletlli&umbn】ln.
;1.4StaticalaI|Forthestati~calcomp~_dsoninPBM- ;NCs.Student’8t-test瑚applied.Theei乎lilicamlevd懈set1o
;0.o5.
;2Results
;2.1SerialchaI1geinthephbloodWBCinre.
;cipientmiceFiveofmiceweretransplantedwitIlC7c ;genetransducedhumanCD34celltIllmtailvein, ;controlledwitIlmicetransplantedwitIlneoRgenetrans. ;ducedCD34cells.A儿tIlemiceweretreatedwitIlcaIb0. ;platin(1.2rag/mouse)intraperitonealyonday28after ;transplantation.Peripheralblood(PB)WBCwerechecked ;onday0,4,7andday14ofcarbeplatinadministration. ;Afterfirstooul’$~ofcar~platin,therewasnosignificant
;differenceinPB—WBCbetween2groups,huttherewas
;significantdifferenceaftertIlesecondeour8~ofcarl~ ;platin.(埏.1).
;0471428323542566O637O
;daysafterlstcourseofcarboplatin.，，
;Fig.1SerialchangeinWBC删aftertmammmtllpla面.
;?
;miceunderwent粥Il0缸III!all碰onwith(;ST-7cle砸眈如ctedhl衄锄
;coldbloodCD34cell;umicellIldeI删xenotransplantationwithneoRgeaetransfeetedh咖
nc0Idb1o0dED34+cells
;2.2GST-~geneexpressioninthebonenlalTOWceilsinalltheGST-~rmice,whileneoRmicedidnotexhi
bitany
;recipientmiceGST=~mRNAwasdetectedintheBidsofGST-~n~NA(fig.2).
;A
;M12345
;~364bp
;M12345
;—7l5bp
;一364bp
;2RT-PQt
;.
;?0f7cmRNAinPBMNCsfromrecipientmice2weksafterthe3rdeomseofcarboplatin.
;A,n~’ansfeeted;B,
;bl一7qtransleeted
;3D缸a
;Theefficacyofmanychemotherapeuticagentsis ;limitedbytheirtoxicitytononTlaltissues ;,
;resultingin
;nalTOWtherapeuticindex.Myelosuppressionisthern0st ;frequentacutedoee-limitingsideeffectresponsibleforde. ;1aysinchemotherapyadministrationord0sealH_e删a！i0ll
;withdecreasedantitumourresponse.na血吼.c0l呦iniI1g
;regiarethecommonlyusedchemotherapeuticregi. ;399
;
;现代妇产科进展2OO3年9月第l2鲞筮塑.Sep.20O3,Vo1.12,No.5 ;ITI~/I$forovariancsIlcer.Dnlgresistancetoplatinumand ;alk)ragentsarel~]8tedtoGST-xgeneexpression? ;GSr-7【gemetransductionmayconferresistanceto ;bematopoieticprogenitors~instthesereagentsandpro—
;tectionofmyelosuppressioninducedbyPCorPAC ;chemotherapy.Invitroexperimenthadbeence~mnedthat ;GSIIletnmsductionintohumanCD34cellscould ;conferdmgresistancetondrhmycinand4一HC(active
;formofcyclophoophamide).Carboplatlnisasecond—gen—
;erationplatinumcomplex.Clinically,carhophtlncansub—
;stitutecisplatininthetreatmentofovariancaIlcer,non—
;smallcellande~1.sivestage锄aU—celllungcancer.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 ;thatofcisphtintogetthes毗neresultf0lrtl1Ietreatmentof
;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-7【genetransfectionintohumancordbloodCD34 ;ceils.
;ThemechanismofdrugresistanceduetoC7cis
;basedonitsability:(i)tocatalyzetheconjugaionof ;electrophilictoxinswithglutatlli0ne,r~ultmSinnllol~po- ;hrandreadilyexcretedmetabolites,(ii)tosequester ;toxinsthmu#hishaffufitybinding,and(iii)toremove ;toxicperoxidestll删曲intrinsic0I学IrIicpemxidaseactivi一
;哆.Ourinvivodabindlca~!tlI砒GST-~geaetrs/lsferre—
;ducedcarboplatin—inducedmDnali哆,s;LI璎;e{|til1gtlI砒nddi—
;tionalchemotherapymaybe西v衄duringtheposttransla—
;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#一dose
;chemotherapies,andtheseIen朔【1soccasionallycontain
;血垮thathavembeariIlg?resistancerelevamtocsI7【.
;1口h,itisnecessarytoccmbimanotherresistance ;genesuchasMDR1andDHFBin池togeneratedru乎re—
;sistal~bonem删inhumancaI妇p日md既lngse—
;quential珊幽pledrugcombinationd目llod1嗍.
;[1]?岣删T,Sal,~ldS,l萨T,eta1.Gsr.,cgemtra嘴-
;ducedhematopoieticprogenitorcell缸:哪印lam锄ovm
;thebonemarIDwtoxicity0f~ydoph~phmid~inmice[J】.
;HumGemTher,2000,11:1671-1681
;[2]SuWC,Q狙ngSL,CheaTY,eta1.c’釉pBri8眦ofinvitro
;growth-inhibitoryactivity0fca~oplmlnandcisp)~_n?
;leukemiceelhandk血越0peti?ptogemlmm:tl~m
;pressiveactivity0fc日曲0l】la血maybee砷erthanits811-
;fileukemieect[J】.】两JOmd,2OOO,30:562-567
;[3]YuLc,W枷DA,Slllwt]_~-E,就a1.Umdatedcordbloodtra嘴.
;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:勰240
;[53StefaniehEG,Cadson-Zenne~CC,McEvoyK,eta1.Dose ;scheduleofrccombin~murin~thro~poietin面0ftomyelo-
;suppressiveand舶吐tbinmlce[J].Cancer
;ClaemotherPharmaed,加01,47:777
;[6]UedaT,TsujiK,YoshinoH,就a1.中dh,m~NOD/
;SClD-repopuht~cellsbystemoellfactor,Flk2/Flt31ir_d, ;~ietln,il-6,andsolubleil-6r?叩[J].’nJQi|I
;Invest.20o0,1O5:1013-1021
;
;