帕金森病脑内移植基因工程细胞系的构建

:250012,();()(PD),(hTH)pcDNA3/hTH,SH2SY5YG418,pcDNA3/hTHSH2SY5Y,SH2SY5YpcDNA3/hTHSH2SY5YhTH,PDTheconformationofcelllinebygeneticengineeringofintracerebraltransplantationinPDYANGYang,WANGXiaomin,WUChengyuan,etal.DepartmentofNeurosurgery,QiluHospital,ShandongUniversity,Jinan250012,ChinaAbstractObjectiveToconstructhumantyrosinehydroxylase(hTH)geneexpressingcell2linefortheexperimentofgenetherapyforParkinsonpsdisease(PD).MethodsRecombinantplasmidpcDNA3/hTHwassub2cloned,purifiedandtransfectedintohumanneuroblastomacell2line(hybridoma)SH2SY5Ycellswithliposomemethod(lipofectin).UnderthepressureofG418(800g/l),drug2resistantcloneswerescreened,separatelycul2turedandassayedimmunocytochemicallytoestablishhTHexpressingclones.ResultsSH2SY5YcellstransfectedwithpcDNA3/hTHwereobviouslystainedimmunocytochemicallyincontrasttothecontrolSH2SY5Ycellswhichweremerelynonspecificallystained.ConclusionsSH2SY5YcellstransfectedwithpcDNA3/hTHcanefficientlyexpresshTH,whichispotentiallyapplicabletonon2humanprimateexperimentsforthetreatmentofPD.KeywordsParkinsonpsdiseaseTyrosinehydroxylaseGenetherapy(PD),,PDpcDNA3/hTHSH2SY5Y,G418,hTH1.pcDNA3/hTH:pcDNA3/hTH,pcDNA3/hTHTOP10,DNA,(PEG),2.(hTH):()2SH2SY5Y,SH2SY5Y,G418():24,G418(GENETICIN)(DMEM),1001000g/ml,100g/ml13105/SH2SY5Y,37CO214,G418()10526/ml4320021181ChinJNeurosurg,January2002,Vol118,No11315cm630%50%4pcDNA3/hTHSH2SY5YLipofectinIBICO48,15G418800g/ml(700g/ml),37,5%CO21457G418,57()1014G418200g/ml,,10l,5101015,253.(hTH)22()TH;:(1):G418,;(2):SH2SY5Y,;(3):SH2SY5Y,24,1.THpcDNA3/hTH::,EcoRI119kb(hTHcDNA)515kb(1),pcDNA3/hTH2.G418:G418()700g/ml3.hTH:,pcDNA3/hTHSH2SY5YTHNo.5,6,7(2)THSH2SY5Y(3),(4),hTHPD2DA1:pcDNA3/hTH;:1:pcDNA3/hTH:EcoRV;2:pcDNA3/hTH:EcoRIM;2:pcDNA3/hTHSH2SY5Ycells3:SH2SY5Y5320021181ChinJNeurosurg,January2002,Vol118,No114:(,)DA,PD80(TH)DA,,DA1989Wolff[1]THcDNA,TH,DA,PD,[2214]TH,,,[9],,[15],,,,,pcDNA3/hTHSH2SY5Y,hTH,PD1WolffJA,FisherLJ,XuL.GraftingfibroblastsgeneticallymodifiedtoproduceL2dopainaratmodelofParkinsondisease.ProcNatlAcadSciUSA,1989,86:901129014.2Uchidak,IshiiA,kanedaN,etal.TetrahydrobiopterindependentproductionofL2dopainNRKfibroblaststransfectedwithtyrosinehyd2toxylasecDNA:futureuseforintracerebralgrafting.NeurosciLett,1990,109:2822286.3HorellorP,BrundinP,KalenP,etal.Invivoreleaseofdopaanddopaminefromgeneticallyengineeredcellsgraftedtothedenervatedratsstriatum.Neuron,1990b,5:3932402.4JiaoS,WolffJA.Long2termsurvivalofautologousmusclegraftsinratbrain.NeurosciLett,1992,137:2072210.5JiaoS,SchultzE,WolffJA.Intracerebraltransplantsofprimarymus2clecells:apotentialplatformfortransgeneexpressioninthebrain.BrainRse,1992,575:1432147.6KawajaMD,GageFH.MorphologicalandneurochemicalfeaturesofculturedprimaryskinfibroblastsofFischer344rat.Followingstratalim2plantation.JCompNeurol,1992,317:1022106.7JiaoS,GurevichV,WolffJA.LongtermcorrectionofratmodelofParkinsonpsdiseasebygenetherapy.Nature,1993,326:4502453.8SuhrST,GageFH.Genetherapyforneurologicdisease.ArchNeurol,1993,50:125221268.9MaysingerD,PiccardoP,LiberiniP,etal.Encapsulatedgeneticallyengineeredfibroblasts:releaseofnervegrowthfactorandeffectsinvivoonrecoveryofcholinergicmarkersafterdevascularizingcorticallesions.NeurochemInt,1993,24:4952503.10MochizukiH,GotoK,MoriH,etal.Histochemicaldetectionofapop2tosisinParkinsonpsdisease.JNeurolSci,1996,137:1202123.11MouradianMM,ChaseTN.GenetherapyforParkinsonpsdisease:Currentknowledgeandfutureperspective.Genetherapy1997,4:506.12..,1994,14:7172719.13,,.GDNF.,1995,27:248.14,,..,1996,12:952102.15RayJ,GageFH.Spinalcordneuroblastsproliferateinresponsetoba2sicfibroblastgrowthfactor.JNeurosci,1994,14:354824564.(:2001208229)6320021181ChinJNeurosurg,January2002,Vol118,No11