人骨髓间充质干细胞分离鉴定方法的改进.doc

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1、人骨髓间充质干细胞分离鉴定方法的改进作者：吴洁莹，廖灿，许遵鹏，陈劲松，辜少玲【摘要】本研究的目的是建立一种基于临床移植需要的分离、培养、鉴定人骨髓间充质干细胞（mesenchymalstemcells，MSC）的方法，为配合造血干细胞移植的临床应用提供实验基础。采用Percoll分离液（1.073g/ml）从新鲜成人骨髓穿刺液中分离MSC，应用贴壁筛选法传代培养，流式细胞仪检测细胞表面抗原，成骨诱导体系（地塞米松0.1μmol/L、β-甘油磷酸钠10mmol/L、抗坏血酸磷酸盐50μmol/L）及脂肪诱导体系（地塞米松1μmol/L、牛胰岛素5mg/L、1-甲基-3-异丁基-黄

2、嘌呤0.5mmol/L、消炎痛60μmol/L）分别诱导MSC定向分化，通过细胞形态观察及免疫化学染色进行鉴定。结果表明：从成人骨髓液中分离培养出MSC，稳定表达CD73、CD105、CD166，不表达CD34、CD45。定向诱导的成骨细胞表达碱性磷酸酶活性，脂肪细胞内出现明显的脂滴。结论：采用密度梯度离心联合贴壁筛选法并通过流式细胞术检测及定向诱导分化，能够从成人骨髓液中分离出MSC，并完成细胞表型及多向分化潜能的初步鉴定。本操作方案具有一定的临床应用价值。【关键词】间充质干细胞　　AmodifiedMethodtoIsolateandIdentifytheAdultMesen

3、chymalStemCellsfromHumanBoneMarrow　　AbstractThestudywasaimedtoestablishaprotocolofisolatingandculturingadultmesenchymalstemcells(MSC)fromhumanbonemarrowaspirateandidentifythembysurfaceantigenanalysisandcommitteddifferentiationinordertoprovideanexperimentalfoundationforachievingatherapeuticben

4、efitinapplyingMSCinhematopoieticstemcelltransplantation.MSCswereobtainedfromfreshhumanbonemarrowaspiratebygradientcentrifugationwithPercoll(1.073g/ml)andanchoringcultureinL-DMEMwith10%fetalbovineserumbyafullmediumexchangeevery3days.TheMSCsurfaceantigens，includingCD34，CD45，CD73，CD105，CD166，wer

5、eanalyzedonFACScanflowcytometer.Undercultureinconditionedmediumforosteogenesis(thehormonecocktailcontaining0.1μmol/Ldexamethasone，10mmol/Lglycerol-2-phosphateand50μmol/Lascorbicacid)andadipogenesis(thecocktailcontaining1μmol/Ldexamethasone，5mg/Linsulin，0.5mmol/L1-methyl-3-isobutylxanthineand6

6、0μmol/Lindomethacin)，MSCscommittedlydifferentiatedintoosteoblastsandadipocytes.Thedifferentiatedmesenchymalstemcellswereidentifiedbymorphological6observationandimmunohistochemicalstaining.Theresultsshowedthatbygradientcentrifugationandadhesionculture，MSCscouldbeisolatedandculture-expandedfrom

7、humanbonemarrowaspirate.ThesecellswereuniformlynegativeforCD34，CD45andpositiveforCD73，CD105andCD166.Theosteogenicdifferentiatedcellswerepositiveforalkalinephosphatase(ALP)andtheadipogenicdifferentiatedcellsdisplayedaccumulationoflipidvacuoles