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《大鼠骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及gfp轉(zhuǎn)染標(biāo)記》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。
1、大鼠骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及GFP轉(zhuǎn)染標(biāo)記發(fā)表吋間：2011-2-1715:31:47來源：創(chuàng)新醫(yī)學(xué)網(wǎng)醫(yī)學(xué)編輯部推薦作者：王亭忠，馬愛群，蔣文慧，徐正云，席雨濤作者單位(西安交通大學(xué)第一I矢院心內(nèi)科;教育部環(huán)境與疾癇相關(guān)基因重點(diǎn)實(shí)驗(yàn)室，陜西西安710061)【關(guān)鍵詞】大鼠，骨髓間充質(zhì)干細(xì)胞;培養(yǎng);綠色熒光蛋白;轉(zhuǎn)染摘要：目的建立大鼠骨髓間充質(zhì)干細(xì)胞(MSCs)的分離和培養(yǎng)方法，檢測(cè)綠色熒光蛋白基因轉(zhuǎn)染MSCs的瞬吋表達(dá)及轉(zhuǎn)染效率。方法應(yīng)用Percoll密度梯度離心法分離人鼠MSCs，貼壁法不斷純化，流式細(xì)胞儀檢測(cè)2代
2、細(xì)胞表而CD34、CD71和CD90的表達(dá)，然后用pEGFPK3與Lipofectamine2000不同濃度比例轉(zhuǎn)染MSCs,熒光倒罝顯微鏡下觀察瞬時(shí)表達(dá)及轉(zhuǎn)染效率。結(jié)果①原代MSCs多呈紡錘形或梭形，宥聚集生長(zhǎng)的傾向，多3?5個(gè)細(xì)胞成為一個(gè)集落。傳代后，細(xì)胞變力形態(tài)均一的排列冇序的成纖維細(xì)胞樣，K梭形，胞漿突起較少，胞體輪廓不甚沾晰，胞體也相對(duì)較火;②GHP轉(zhuǎn)染后24h即可見綠色熒光蛋白表達(dá)，72h表達(dá)最強(qiáng)，此只逐漸減弱，到4周吋仍可見少量表達(dá);③轉(zhuǎn)染效率與質(zhì)粒和脂質(zhì)體的濃度比例有關(guān)，1:2到1:3最髙。結(jié)論利用Per
3、coll密度梯度離心法結(jié)合貼壁法町以獲得比較純的MSCs;GFP轉(zhuǎn)染是一種較好的MSCs鈿記方法。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
