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1、·482·ChineseJournalofReparativeandReconstructiveSurgery,May2007,Vol.21,No.5人胶质源性神经生长因子真核表达载体构建及在大鼠脊髓组织中的表达李琦曾炳芳徐建广孔维清【摘要】目的探索人胶质源性神经生长因子(humanglialderivedneurotrophicfactor,hGDNF)真核表达载体,体内直接转染SD大鼠脊髓组织治疗急性脊髓损伤(spinalcordinjury,SCI)的可能性。方法基因重组和限制性内切酶酶切构建真核表达载体pcDNA32hGDNF,经脂质体DOTAP
2、介导质粒转染SD大鼠脊髓组织,作为实验组;对照组大鼠直接注射空载体脂质体混合物。14d后取材,RT2PCR及Westernblot检测hGDNFmRNA及蛋白表达。结果重组真核表达载体pcDNA32hGDNF经限制性内切酶HindIII和Xba2É酶切后,电泳显示400bphGDNF目的片段和5400bppcDNA3载体片段。转染大鼠脊髓组织后14d,RT2PCR检测出目的基因mRNA,Westernblot检测出hGDNF的蛋白表达。结论构建的真核表达载体pcDNA32hGDNF能在转染的大鼠脊髓组织中表达,可为基因治疗修复急性SCI奠定基础。【关键词
3、】脊髓损伤胶质源性神经生长因子基因转染脂质体大鼠中图分类号:R318R65112文献标识码:ACONSTRUCTIONOFEUKARYOTICEXPRESSIONVECTORFORHUMANGLIALDERIVEDNEUROTROPHICFACTORANDITSEXPRESSIONINSPINALCORDTISSUEOFSDRATöLIQi,ZENGBingfang,XUJianguang,etal.DepartmentofOrthopedics,theSixthPeople'sHospital,ShanghaiJiaotongUniversity,Sh
4、anghai,200233,P.R.China.E2mail:drlee12@126.comCorrespondingauthor:ZENGBingfang,E2mail:bingfangz@yahoo.com.cn【Abstract】ObjectiveToinvestigatethepossibilityofconstructingeukaryoticexpressionvectorforhumanglialderivedneurotrophicfactor(hGDNF),transfectingittospinalcordtissueofratsso
5、astotreatacutespinalcordinjury.MethodsTheeukaryoticexpressionvectorpcDNA32hGDNFwasconstructedbyrecombinantDNAtechnique,transfectedintoglialcellandneuronofspinalcordbyliposomeDOTAPasexperimentalgroup.Incontrolgroup,mixtureofemptyvectorandliposomewasinjected.ThemRNAandproteinexpres
6、sionsofhGNDFweredetectedbyRT2PCRandWesternblot.ResultsAftertherecombinanteukaryoticexpressionvectorforhGDNFwasdigestedwithHindIIIandXba2É,electrophoresisrevealed400bpfragmentforhGDNFgeneand5400bpfragmentforpcDNA3vector.Inthetransfectedspinalcordtissue,themRNAandproteinexpressions
7、ofhGDNFgeneweredetectedwithRT2PCRandWesternblot.ConclusionTheconstructedeukaryoticexpressionvectorpcDNA32hGDNFcouldbeexpressedinthetransfectedspinalcordtissueofrat,soitprovidebasisforgenetherapyofacutespinalcordinjury.【Keywords】SpinalcordinjuryGlialderivedneurotrophicfactorGenetr
8、ansfectionLiposomeRats脊髓损伤(spinalcordinj
