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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。
Objective To observe the biological characteristics of human bone marrow mesenchymal stem cells (hMSCs) cultured in vitro and to explore the methods of transdifferentiating them into neural precursor cells (NPCs). Methods Human bone marrow mesenchymal stem cells (MSCs) were isolated by density gradient centrifugation and adherent method. Cell morphology, growth, surface markers, osteogenic and cartilage formation and adipogenesis were observed. The third generation of cells were selected for induction. The cells were induced to proliferate by embryonic stem cell culture medium and then induced by neural induction with 5-azacytidine and trichostatin A. Seven days later, some samples were subjected to Nestin and Sox2 immunofluorescence staining And RT-PCR. The other samples were cultured in B27-containing neuroblastoma medium for 7 days, and then NF-L was detected by immunofluorescence. Results The purity of hMSCs isolated and cultured was high, the positive rates of CD29 and CD44 were above 90%, and the ability of osteogenic, cartilage and adipogenesis was obvious. After treated by 5-azacytidine and trichostatin A, Differentiation into neural progenitor cells, immunofluorescence staining and RT-PCR results showed that the induced cells could express Nestin and Sox2, which were the precursors of neural precursor cells. After culturing in neural culture medium for detection of neural cell marker NF- L, showing more positive cells. Conclusion hMSCs can be isolated, cultured and expanded in vitro. After being modified by drugs, hMSCs have the potential to differentiate into neural progenitor cells.