人骨髓间充质干细胞的扩增及向成软骨样细胞的诱导分化（完整版）实用资料(可以直接使用，可编辑完整版实用资料，欢迎下载）人骨髓间充质干细胞的扩增及向成软骨样细胞的诱导分化邱丽燕,王金福(浙江大学生命科学学院浙江杭州310012摘要从人骨髓中分离和培养间充质干细胞,在原代和传代培养中其形态学上均为贴壁、纤维状的细胞,随着代次的增加细胞形态更加趋于一致。流式细胞仪鉴定CD45、CD34、CD117、HLA-DR阴性,CD29、CD166阳性,并随着培养的MSCs代次的增加,阳性比率和阴性比率出现相应的上升和降低,说明细胞纯度增加。第三代的MSCs大约有80%处于G0/G1期,S+G2+M期约20%左右,说明MSCs有强大的增殖潜能。MSCs在传代培养中的最适接种密度约为5.0×103/cm2左右。MSCs在体外经历了滞缓期、对数生长期、平稳期三个时期,传代培养的MSCs生长速度比原代的MSCs明显快很多。采用程序降温法对MSCs进行冻存,60~90天后对冻存的MSCs进行复苏,发现MSCs仍可在体外良好生长4~6代,特定的MSCs样本可以扩增10代,说明经过冻存的细胞仍具有良好的增殖能力。对四例标本的前三代MSCs在体外利用TGF–β1和维生素C诱导两周后,用RT-PCR证明MSCs表达软骨特异性的II型前胶原的mRNA,说明我们培养的细胞具有向成软骨样细胞分化的潜能,由此进一步证实其为MSCs。关键词骨髓间充质干细胞扩增成软骨样细胞ExpansionandChondrogenicInductionofHumanBoneMarrowMesenchymalStemCellsQiuLiyan,WangJinfu(CollegeofLifeScience,ZhejiangUniversity,Hangzhou,Zhejiang310012AbstractMSCsfromhumanBMwereisolatedbyFicolldensitycentrifugationandculturedexpandedinDMEM-LGcontainingFBS.MSCsareadherentandfibroblastic,andmaintainedsimilarmorphologywithpassaging.FlowcytometryanalysisindicatedthatMSCswereuniversallypositiveforCD29,CD166,andnegativeforCD34,CD45,CD117,andHLA-DR.Atpassage3,cellcyclestatusanalysisbymeasuringDNAcontentrevealedthatasmallpopulationofthecellswasengagedinproliferation(S+G2+M=20%,andmorethan80%ofcellswereinG0/G1phase.ThenumberofcellsinG0/G1phasedecreasedwithpassage.Inpassageculture,Itisproperforthecellsplatedatthedensityof5000cells/cm2topropagate.TheMSCsofpassage1to3werefrozeinliquidnitrogenbytwo-stepfrozenprocedure,after60~90days,wethawedthefrozenMSCsandfoundthecellscanproliferateabout4~6passagesinvitro,10passagesespeciallyforsample17.ItsuggestedthatthefrozenMSCsstillhavegoodexpansionability.Atpassage1to3,fourcaseswasinducedbythecombinationofTGF-β1andVitamineCinvitrofortwoweeksandexpressedarticularcartilagematrix-procollagenIImRNA.KeywordMesenchymalstemcells(MSCs,expansion,chondrocyte骨髓间充质干细胞(mesenchymalstemcells,MSCs是存在于骨髓中的一类多能干细胞。Friedenstein等[1]首先发现MSCs在体外可分化为骨和软骨组织,即使经过20-30次细胞分裂后,其分化特性也不会消失。Pittenger等[2]通过扩增单个MSCs的克隆后,在不同的条件下诱导其分化,结果分别分化出骨,软骨,脂