【摘要】 目的观察体外培养的人骨髓间充质干细胞(hMSCs)的生物学特性,并探讨使其转分化为神经前体细胞(NPCs)的方法。方法以密度梯度离心和贴壁法相结合分离成人骨髓间充质干细胞,并观察细胞形态、生长、表面标记以及成骨和成软骨及成脂肪能力的情况。选用第3代细胞进行诱导,先经胚胎干细胞培养液扩增,再用加有5-氮胞苷和曲古菌素A的神经诱导液诱导,7d后,一部分样本进行Nestin、Sox2免疫荧光染色和RT-PCR检测;另一部分样本在含有B27的神经培养液中继续培养7d,然后进行NF-L的免疫荧光检测。结果分离培养的hMSCs纯度较高,CD29、CD44的阳性率均在90%以上;具有明显的成骨、成软骨和成脂肪能力;经5-氮杂胞苷和曲古菌素A作用后能向神经前体细胞分化,免疫荧光染色及RT-PCR结果显示,诱导后的细胞能特异性表达神经前体细胞标志物Nestin和Sox2;在神经培养液中继续培养后检测神经细胞标记物NF-L,可见较多阳性细胞。结论hMSCs可在体外进行分离培养扩增,经药物修饰后具有向神经前体细胞分化的潜能。更多还原

【Abstract】 Objective To investigate biological characteristics of cultured human bone marrow mesenchymal stem cells（hMSCs） in vitro,and differentiation potential to neural precursor cells（NPCs）.Methods hMSCs were isolated with density gradient centrifugation and cell adhesion from adult bone marrow.The cell morphology,growth,surface markers were investigated,and osteogenic,chondrogenic,adipogenic differentiation potential were analyzed as well.At the 3 passage,hMSCs were expanded by embryonic stem cell maintenance medium,then these cells were exposed to neural induction medium,that contained 5-aza-2′-deoxycytidine（5azadc） and Trichostatin A（TSA）.After 7 days,half of the samples were detected the markers for multipotent NPCs,Nestin and Sox2 by immunofluocence and RT-PCR.Further,another half of the samples were grown an additional week in Neurobasal/B₂₇,to observe to differentiate into neural phenotypes.Results For acquired hMSCs,the positive ratio of CD₂₉,CD₄₄ were all above 90%;The osteogenic,chondrogenic and adipogenic differentiation potential were detected obviously;hMSCs induced by 5azadc and TSA could be identified by the positive staining for Nestin and Sox2,RT-PCR assessment showed that the differentiated cells expressed Nestin and Sox2.And after 1 week in Neurobasal/B₂₇,hMSCs differentiated into NF-L immunopositive cells with similar neuronal morphology.Conclusion hMSCs can be cultured and expanded in vitro;after treated with epigenetic modifiers agents in neural environment,hMSCs efficiently are differentiated into NPCs.更多还原