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1、大鼠骨髓间充质干细胞的分离培养及GFP转染标记发表吋间：2011-2-1715:31:47來源：创新医学网医学编辑部推荐作者：王亭忠，马爱群，蒋文慧，徐正云，席雨涛作者单位(西安交通大学第一I矢院心内科;教育部环境与疾痫相关基因重点实验室，陕西西安710061)【关键词】大鼠，骨髓间充质干细胞;培养;绿色荧光蛋白;转染摘要：目的建立大鼠骨髓间充质干细胞(MSCs)的分离和培养方法，检测绿色荧光蛋白基因转染MSCs的瞬吋表达及转染效率。方法应用Percoll密度梯度离心法分离人鼠MSCs，贴壁法不断纯化，流式细胞仪检测2代
2、细胞表而CD34、CD71和CD90的表达，然后用pEGFPK3与Lipofectamine2000不同浓度比例转染MSCs,荧光倒罝显微镜下观察瞬时表达及转染效率。结果①原代MSCs多呈纺锤形或梭形，宥聚集生长的倾向，多3〜5个细胞成为一个集落。传代后，细胞变力形态均一的排列冇序的成纤维细胞样，K梭形，胞浆突起较少，胞体轮廓不甚沾晰，胞体也相对较火;②GHP转染后24h即可见绿色荧光蛋白表达，72h表达最强，此只逐渐减弱，到4周吋仍可见少量表达;③转染效率与质粒和脂质体的浓度比例有关，1:2到1:3最髙。结论利用Per
3、coll密度梯度离心法结合贴壁法町以获得比较纯的MSCs;GFP转染是一种较好的MSCs钿记方法。RatmesenchymalstemcollscultureandlabelwithgreenfluorescentproteingenetransfectionUangTingzhong,MaAiqun,JiangWenhui,XuZhengyun,XiYutao(DepartmentofCardiology,FirstHospitalofXianJiaotongUniversity,KeyLaboratoryofEnvi
4、ronmentandGenesRelatedtoDiseasesofMinistryofEducation,Xian710061,China)ABSTRACT:ObjectiveToestablishamethodofisolationandcultureoftheratmesenchymalstemcells(MSCs),andtoinvestigatethetransientexpressionofgreenfluorescentproteingenetransfectionintoMSCs.MethodsMSCs,
5、whichwereinitiallyisolatedfromthebonemarrowofrats,wereculturedinvitroandtestedbyflowcytomertry.ThentheyweretransfectedwithpEGFPN3bymeansofLipofectamine2000.TheratioofplasmidtoLipofectaminevariedaccordingtotheexperimentdesign.TheresultsofthotransientexpressionofGF
6、Pandtransfectionefficiencywereobservedbyfluorescencemicroscope.ResultsMSCswereadherent,elongated,andspindleshapedintheprimarycultureafter24hoursofplating,andthenbecameseverallittleclones;Duringmitosisthecellsregainedaroundedappearance,andremainedlooselyattachedun
7、tildivisionwascompleted.Whentheyreached80%confluence,MSCsweresubculturedandgaineduniformshapeandsimilargrowthpattern.TheexpressionofGFPbeganat24hoursaftertransfection,andreachedthepeakat72hoursanddecreasedin1week;however,thetransientexpressionremainedformorethan4
8、weeks.Transfectionefficiencywascorrelatedwiththeratioofplasmidtolipofectamine.ConclusionHighpuredMSCscanbeobtainedbyPercollgradientcentrifugingandadherentmetho
