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1、人骨髓間充質(zhì)干細(xì)胞體外向肝樣細(xì)胞的誘導(dǎo)分化【摘要】目的：研究人骨髓來(lái)源的間充質(zhì)干細(xì)胞(humanbonemarrowderivedmesenchymalstemcells，hBM-MSCs)在肝細(xì)胞生長(zhǎng)因子(HGF)、成纖維細(xì)胞生長(zhǎng)因子-4(FGF-4)、抑瘤素M(OSM)誘導(dǎo)下，體外向肝樣細(xì)胞的誘導(dǎo)分化。方法：通過(guò)密度梯度離心和貼壁培養(yǎng)法獲得hBM-MSCs，分離的hBM-MSCs擴(kuò)增傳至第6代，流式細(xì)胞儀檢測(cè)hBM-MSCs表面CD34、CD45、CD105、CD166表達(dá)。第6代hBM-MSCs生長(zhǎng)達(dá)100%融合時(shí)采用兩步法對(duì)
2、hBM-MSCs進(jìn)行誘導(dǎo)分化。第1～第14天采用IMDM誘導(dǎo)培養(yǎng)基(含2%FCS、20ng/mLHGF、20ng/mLFGF-4)，第15～第21天在上述培養(yǎng)基中加OSM(10ng/mL)，促進(jìn)誘導(dǎo)細(xì)胞的成熟，同時(shí)收集第0、第7、第14、第21天培養(yǎng)上清液標(biāo)本，ELISA法測(cè)ALB濃度。誘導(dǎo)培養(yǎng)21d后采用RT-PCR、免疫熒光技術(shù)檢測(cè)誘導(dǎo)后肝細(xì)胞特異標(biāo)志CK18、ALB的表達(dá)，PAS染色檢測(cè)誘導(dǎo)MSCs糖原儲(chǔ)存。結(jié)果：傳至第6代的hBM-MSCsCD34、CD45表達(dá)陰性，CD105、CD166表達(dá)陽(yáng)性。在細(xì)胞因子的作用下，經(jīng)過(guò)2
3、1d的體外培養(yǎng)，hBM-MSCs形態(tài)發(fā)生變化，由長(zhǎng)梭形變?yōu)槎噙呅巍㈩悎A形，并具有肝細(xì)胞特異的表型和功能。RT-PCR可檢測(cè)到CK18、ALB表達(dá)，免疫熒光可檢測(cè)到誘導(dǎo)后細(xì)胞內(nèi)CK18、ALB，培養(yǎng)上清中有ALB的分泌，且誘導(dǎo)后的肝樣細(xì)胞內(nèi)有糖原儲(chǔ)存。結(jié)論：hBM-MSCs體外能轉(zhuǎn)化為具有肝功能的肝樣細(xì)胞。15【關(guān)鍵詞】間充質(zhì)干細(xì)胞;人骨髓間充質(zhì)干細(xì)胞;肝樣細(xì)胞;細(xì)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
