人脐带间充质干细胞的分离培养及向成骨成脂分化的实验研究.doc

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1、人脐带间充质干细胞的分离培养及向成骨成脂分化的实验研究作者：孙国栋，李志忠，王晶，林永新，洪亮，吴波文，焦根龙,邵建立【摘要】目的探讨并优化人脐带间充质干细胞(hUCMSCs)体外获取及培养增殖的方法并鉴定；诱导其向成骨细胞和脂肪细胞分化，为其进一步应用奠定基础。方法用酶消化法分离培养hUCMSCs,通过传代培养、扩增，倒置光学显微镜及原子力显微镜下观察细胞形态；MTT法检测细胞增殖曲线，流式细胞仪检测免疫表型；体外向成骨、脂肪方向诱导分化，并通过特异性染色鉴定其分化能力。结果采用酶消化法能有效分离纯化hUCMSCso接种24h,贴壁细胞形态多为长梭型、多边形或成纤维

2、细胞样形态，大小均一。P3、P7代细胞的生长曲线基本一致，增殖能力强。流式细胞仪分析，第3代细胞强烈表达CD29、CD44、CD105,不表达CD34、CD45和HLADR。经成骨诱导分化后4周，碱性磷酸酶染色呈强阳性，茜素红染色可见明显钙结节；经成脂诱导3周，油红0染色呈阳性，有明显的脂滴出现。结论本实验建立了一套体外稳定培养扩增hUCMSCs的方法。所培养的细胞成分单一、扩增迅速、生物学形状稳定，并能使其在体外诱导向成骨细胞和脂肪细胞分化，有望成为组织工程较为理想的种子细胞。【关键词】脐带；间充质干细胞；分化；成骨细胞；脂肪细胞ABSTRACT:Objective

3、Toestablishanoptimizedmethodtoisolate,cultureandidentifyhumanumbilicalcordmesenchymalstemcells(hUCMSCs)inviti'o&ndinducetheii'osteogenicandadipogeniedifferentiation.MethodsThehUCMSCswereisolatedfromhumanumbi1icalcordbydigestionwithcollagenase・Afterserialsubcultivationinvitro,thestemcel1

4、swerepassaged・MorphologicappearanceofhUCMSCswasobservedunderanopticalmicroscopeandatomicforcemicroscope・TheproliferationratewasmeasuredbyMTTassay.Cellcycleandsurfaceantigensweremeasuredbyf1oweytometry.Theosteogenicandadipogeniedifferentiationwastestedandevaluatedbyspecificstainingmethod

5、s・Resu1tsTheisolationofhUCMSCsbydigestionwitheol1agenasewasefficient.Afterseededfor24hours,thea.dherentcellsshowedspindlesha.peandfibroblastcell1ikeshapeandthesizeofhUCMSCswashomogeneous・Thesimi1argrowthcurvesofpassage3and7exhibitedagreatpotentialforproliferation.Flowcytometryanalysisre

6、vealedthatCD29,CD44andCDl05werehighlyexpressedonthesurfaceofpassages3cells,buttheexpressionwasnegativeforCD34,CD45andHLADR.Aftercultureininducingmediuni,thecel1sweresuccessful1yinducedintoosteogenicandadipogeniclineages・Thesecellswerehighlypositiveforalkalinephosphatestainingandalsoshow

7、edmineralizationpresentedwithvonkossastainingafter4weekscultureinductionofosteogenicdifferentiation.Furthennore,1iquidvacuolesweredetectedbyoilred0stainingafter3weekscultureinductionofsdipogenicdifferentistion.ConclusionAninvitromethodforisolationandpurificationofhUCMSCsfromhui