BrdU--CFSE和GFP标记大鼠间充质干细胞的比较.docx

BrdU,CFSE和GFP标记大鼠间充质干细胞的比较作者：付霞霏，何援利，杨芳，彭冬先，刘木彪【摘要】目的:研究BrdU,CFSE和GFP作为大鼠间充质干细胞标记物的可行性.方法:分别用上述三种标记物标记大鼠间充质干细胞，检测即时、15和30d的标记率，通过检测细胞周期、凋亡率和描绘生长曲线，来明确标记物对体外培养大鼠间充质干细胞生长特性的影响；通过流式细胞仪检测标记物对细胞表面标志表达的影响.结果:BrdU,CFSE,GFP的即时标记率为%,%,%，1mo后分别下降至%,%,%.三者对体外培养的大鼠间充质干细胞的生长特性和表面标志的表达均无明显影响.结论:GFP,BrdU和CFSE均可以作为大鼠间充质干细胞的标记物.【关键词】绿色荧光蛋白；溴脱氧尿嘧啶核苷；荧光染料CFSE；间充质干细胞【Abstract】AIM:ToinvestigatethefeasibilityoflabelingofBrdU,CFSEandGFPaslabelsofratmesenchymalstemcells(MSCs)andtoinvestigatetheinfluenceofthe3markersonthegrowthcharacteristicsandexpressionsofsurfacemarkersofratMSCs.METHODS:WistarratsbonemarrowderivedMSCswereisolatedandpurifiedinvitrobyPercolldensitygradientcentrifugationcombinedwithadherentmethodandwerelabeledwithBrdU,CFSEandGFP,respectively.Theimmediate,15dayand30daylabelingefficiencyofthe3markerswasquantified.ThelabelingefficiencyofBrdUwasdetectedwithimmunocytochemicalmethod,andthatofCFSEandGFPwasdetectedviaflowcytometry(FCM).ThelabeledMSCswereobservedundermicroscope.CellcycleandapoptosisrateoflabeledMSCswererevealedandthegrowthcurvesweredelineated,soastoidentifywhetherornotthemarkerscastedaninfluenceonthecellgrowthcharacteristicsinvitro.Furthermore,thesurfacemarkers(CD29,CD34,CD44,CD45)ofthelabeledMSCsweredetectedthroughflowcytometryandwerecomparedwiththoseofnonlabeledMSCs.RESULTS:TheimmediatelabelingefficiencyofBrdU,CFSE,andGFPwas%,%,%respectively,andtheefficiencydecreasedto%,%,and%respectivelyafter30d.TherewasnostatisticaldifferenceincellcycleandapoptosisratiobetweenlabeledMSCsandnonlabeledMSCs.ThegrowthcurvesoflabeledMSCsweresimilartothatofnonlabeledMSCs.The3markersdidntinfluencetheexpressionsofsurfacemarkers(CD29,CD34,CD44,CD45)ofratMSCs.CONCLUSION:The3markershavenoeffectsonthegrowthcharacteristicsandexpressionsofsurfacemarkersofratMSCsculturedinvitro.BrdU,CFSEandGFPcanserveaslabelingmarkersofratMSCs.BrdUandCFSElabelingcanbeusedforshorttimetracing,andGFPlabelingforlongtimetracing.【Keywords】GFP;BrdU;CFSE;MSCs0引言骨髓间充质干细胞是定居于骨髓微环境中的一种成体干细胞.MSCs不仅能分泌多种细胞因子调节造血细胞的增