人骨髓间充质干细胞体外向肝样细胞的诱导分化

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1、人骨髓间充质干细胞体外向肝样细胞的诱导分化【摘要】目的：研究人骨髓来源的间充质干细胞(humanbonemarrowderivedmesenchymalstemcells，hBM-MSCs)在肝细胞生长因子(HGF)、成纤维细胞生长因子-4(FGF-4)、抑瘤素M(OSM)诱导下，体外向肝样细胞的诱导分化。方法：通过密度梯度离心和贴壁培养法获得hBM-MSCs，分离的hBM-MSCs扩增传至第6代，流式细胞仪检测hBM-MSCs表面CD34、CD45、CD105、CD166表达。第6代hBM-MSCs生长达100%融合时采用两步法对

2、hBM-MSCs进行诱导分化。第1～第14天采用IMDM诱导培养基(含2%FCS、20ng/mLHGF、20ng/mLFGF-4)，第15～第21天在上述培养基中加OSM(10ng/mL)，促进诱导细胞的成熟，同时收集第0、第7、第14、第21天培养上清液标本，ELISA法测ALB浓度。诱导培养21d后采用RT-PCR、免疫荧光技术检测诱导后肝细胞特异标志CK18、ALB的表达，PAS染色检测诱导MSCs糖原储存。结果：传至第6代的hBM-MSCsCD34、CD45表达阴性，CD105、CD166表达阳性。在细胞因子的作用下，经过2

3、1d的体外培养，hBM-MSCs形态发生变化，由长梭形变为多边形、类圆形，并具有肝细胞特异的表型和功能。RT-PCR可检测到CK18、ALB表达，免疫荧光可检测到诱导后细胞内CK18、ALB，培养上清中有ALB的分泌，且诱导后的肝样细胞内有糖原储存。结论：hBM-MSCs体外能转化为具有肝功能的肝样细胞。15【关键词】间充质干细胞;人骨髓间充质干细胞;肝样细胞;细胞因子　　Abstract:Objective:Toinvestigatethedifferentiationandinductionabilityofhumanbonem

4、arrowderivedmesenchymalstemcellsintohepatocyte-likecellsinducedbyhepatocytegrowthfactor，fibroblastgrowthfactor-4andoncostatinMinvitro.Methods:Mesenchymalstemcells(MSCs)fromhumanbonemarrowwereselectedwithdensitygradientcentrifugationandplasticadherence.Flowcytometrywasp

5、erformedusingthefollowingmouseanti-humanantibodies:anti-CD34FITC，anti-CD45FITC，anti-CD105PE，anti-CD166APC.ThesixthgenerationMSCswereincubatedwiththeeachantibodyandanalyzedwithaFACS-CaliburwithCellQuestsoftware.Forhepatogenicdifferentiation，thesixthgenerationcellsreachi

6、ng100%confluencewereinducedby2-stepmethed.DifferentiationwasinducedbytreatingsixthgenerationMSCswithStep-1differentiationmedium，consistingofIMDMsupplementedwith2%FCS，20ng/mLHGFand20ng/mLFGF-4for14days，followedbytreatmentwithstep-2maturationmedium，consistingofIMDMsupple

7、mentedwith2%FCS，20ng/mLHGF，20ng/mLFGF-4，10ng/mLoncostatinM.Mediumchangeswere15performedtwiceweekly.After21daysculture，mRNAofALBandCK18wasassessedwithreversetranscriptionpolymerasechainreaction.ProductionofALBandCK18wasassessedwithimmunofluorescenceanalysisandElisa.Glyc

8、ogendepositswerevisualisedincellsfixedinparaffinwithconventionalperiodicacid-Schiff(PAS)staining.Results:Thesixthgene