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目的探讨人脐血单个核细胞(UBC-MNCs)体外分离培养和定向诱导分化为神经干细胞(NSCs)的机制,观察培养细胞增殖分化情况,检测NSCs中Foxg1和Nestin基因mRNA的表达情况及相互关系。方法从脐血中分离出UBC-MNCs,用含人表皮细胞生长因子(hEGF)、碱性成纤维细胞生长因子(bFGF)和B27因子的Neurobasal培养基联合诱导其向NSCs方向分化,观察NSCs增殖分化及形态学特点;免疫组织化学法检测培养细胞中神经细胞标志抗原巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的表达;5-溴-2-脱氧尿嘧啶核苷(Brdu)标记靶细胞,并测定增殖指数;RT-PCR法检测诱导前、培养3d、6d、9d、12d细胞中Foxg1和Nestin基因mRNA的表达变化。结果 UBC-MNCs可定向诱导分化为神经元样细胞,表达Nestin、NSE、GFAP标记抗原。Brdu标记细胞阳性率达90%。Foxg1mRNA在诱导前细胞中低表达,诱导后逐渐升高(P<0.05),6d达高峰,后逐渐下降(P<0.05);NestinmRNA表达逐渐升高(P<0.05)。结论体外诱导培养可获得UBC-MNCs源性NSCs,表达神经细胞特异性标志物Nestin、NSE和GFAP蛋白,Foxg1和Nestin基因表达明显增强,是NSCs增殖分化的关键调控因子。脐血能够成为NSCs的新来源。
OBJECTIVE: To investigate the mechanism of human umbilical cord blood mononuclear cells (UBC-MNCs) isolated and cultured in vitro and differentiate into neural stem cells (NSCs) in vitro. To observe the proliferation and differentiation of cultured cells and to detect the expression of Foxg1 and Nestin mRNA and their relationship . Methods UBC-MNCs were isolated from umbilical cord blood and induced to differentiate into NSCs by using Neurobasal medium containing human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF) and B27, and the proliferation of NSCs was observed The expression of Nestin, NSE and GFAP in cultured cells was detected by immunohistochemical method. The expression of 5-bromo- The target cells were labeled with Brdu and the proliferation index was determined. The mRNA expression of Foxg1 and Nestin were detected by RT-PCR before and 3 days, 6 days, 9 days and 12 days. Results UBC-MNCs could be induced to differentiate into neuron-like cells and express Nestin, NSE and GFAP-labeled antigens. Brdu labeled cells positive rate of 90%. The expression of Foxg1mRNA was low in pre-induction cells, and gradually increased after induction (P <0.05). The expression of Foxg1mRNA peaked at 6d and then decreased gradually (P <0.05). The expression of Nestin mRNA increased gradually (P <0.05). CONCLUSION: UBC-MNCs-derived NSCs can be obtained in vitro. Nestin, NSE and GFAP proteins expressing neural cell-specific markers can be obtained. The expression of Foxg1 and Nestin is significantly increased, which is a key regulator of proliferation and differentiation of NSCs. Cord blood can become a new source of NSCs.