Objective To investigate the effect of xenogeneic bone on induced differentiation of neural stem cells (NSCs) .Methods NSCs were isolated from cerebral cortex of the frontal lobe in 15 d rat embryos and were cultured u-sing the neurosphere culture system.The basic culture medium was DMEM/F12,added with 2%B27,20μg/L EGF,20μg/L bFGF and others.After 3 passages,NSCs were stained by Nestin and identified by immunocytochemistry.They were then co-cultured with xenogeneic bone extracts at concentrations of 0.025,0.05,0.1,0.2 g/mL.Results Cells after passage exhibited characteristics of NSCs.Positive expression of microtubule-associated protein 2,glial fibrillary acidic protein and O4 were detected in NSCs co-cultured with 0.2 g/mL xenogeneic bone extracts.That is to say,xenogeneic bone extracts could induce NSCs differentiate into neurons,astrocytes and oligodendrocyte.When the concentration of xe-nogeneic bone extracts was 0.1 g/mL,the proportion of NSCs that differentiate into oligodendrocytes was the most.When the concentration was 0.05 g/mL,the proportion of NSCs that differentiate into astrocytes was the most.Conclusion Xe-nogeneic bone can induce NSCs to differentiate into neurons and glial cells.The proportion of differentiated cells was dif-ferent at various concentrations of xenogeneic bone.Therefore,in its clinical application,the xenogeneic bone combined with certain growth factors to induce NSC differentiation can help repair the damage of the nervous system.%目的 探讨异种骨对大鼠神经干细胞(NSCs)诱导分化的作用.方法 从15 d胎龄大鼠的大脑皮质分离获得NSCs后,以神经球悬浮培养法,采用DMEM/F12为基础培养基,加入2%B27、20 μg/L EGF和20 μg/L bFGF等进行培养.传代3次后,采用巢蛋白染色,免疫荧光细胞化学法鉴定NSCs.然后,加入浓度分别为0.025、0.05、0.1、0.2 g/mL的异种骨浸提液与NSCs进行共培养.结果 传代后的细胞表现出NSCs的特性.与0.2 g/mL异种骨浸提液共培养的NSCs微管相关蛋白、胶原纤维酸性蛋白和O4均呈阳性表达,即异种骨浸提液可显著诱导NSCs分化为神经元、星形胶质细胞和少突胶质细胞;其中异种骨浸提液浓度为0.1 g/mL时,诱导NSCs分化为少突胶质细胞的比例最高;浓度为0.05 g/mL时,分化为星形胶质细胞的比例最高.结论 异种骨具有诱导NSCs分化为神经元及胶质细胞的作用,且浓度不同,诱导分化的细胞比例成分亦不同.临床应用中,可结合一定的生长因子,诱导其分化方向,从而有利于修复神经系统损伤.
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