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ID:15199203
大小:42.00 KB
页数:14页
时间:2018-08-01
《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》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
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hUCMSCs。接种24h,贴
2、壁细胞形态多为长梭型、多边形或成纤维细胞样形态,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O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
3、质干细胞;分化;成骨细胞;脂肪细胞14 ABSTRACT:ObjectiveToestablishanoptimizedmethodtoisolate,cultureandidentifyhumanumbilicalcordmesenchymalstemcells(hUCMSCs)invitroandinducetheirosteogenicandadipogenicdifferentiation.MethodsThehUCMSCswereisolatedfromhumanumbilicalcordbydigestionwithcol
4、lagenase.Afterserialsubcultivationinvitro,thestemcellswerepassaged.MorphologicappearanceofhUCMSCswasobservedunderanopticalmicroscopeandatomicforcemicroscope.TheproliferationratewasmeasuredbyMTTassay.Cellcycleandsurfaceantigensweremeasuredbyflowcytometry.Theosteogenicanda
5、dipogenicdifferentiationwastestedandevaluatedbyspecificstainingmethods.ResultsTheisolationofhUCMSCsbydigestionwithcollagenasewasefficient.Afterseededfor24hours,theadherentcellsshowedspindleshapeandfibroblastcelllikeshapeandthesizeofhUCMSCswashomogeneous.Thesimilargrowth
6、curvesofpassage3and7exhibitedagreatpotentialforproliferation.FlowcytometryanalysisrevealedthatCD29,CD44andCD105werehighlyexpressedonthesurfaceofpassages3cells,buttheexpressionwasnegativeforCD34,CD45andHLADR.Aftercultureininducingmedium,thecellsweresuccessfullyinducedint
7、oosteogenicandadipogeniclineages.Thesecells14werehighlypositiveforalkalinephosphatestainingandalsoshowedmineralizationpresentedwithvonkossastainingafter4weekscultureinductionofosteogenicdifferentiation.Furthermore,liquidvacuolesweredetectedbyoilredOstainingafter3weeksc
8、ultureinductionofadipogenicdifferentiation.ConclusionAninvitromethodforisolationandpurificationofhUCMSC
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