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目的探讨人脐血单个核细胞(UBC-MNCs)体外定向诱导分化为神经干细胞(NSCs)的可行性,观察NSCs中Nestin基因mRNA的表达情况。方法采用密度梯度离心法从人脐血中分离出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)的表达情况;RT-PCR法检测诱导前后培养细胞中NestinmRNA的表达变化。结果 UBC-MNCs可定向诱导分化为神经元样细胞,形成典型NSCs克隆团,表达Nestin、NSE、GFAP标记抗原蛋白,诱导前细胞Nestin、NSE、GFAP的表达均非常低,诱导6d时Nestin阳性细胞数达高峰,然后开始下降,NSE、GFAP阳性细胞数逐渐增高。Brdu标记靶细胞阳性率达90%。Nestin mRNA在诱导前细胞中低表达,诱导3d、6d、9d、12d时表达逐渐升高(P<0.05)。结论体外诱导培养可获得UBC-MNCs源性NSCs,Nestin基因是NSCs增殖分化的关键调控因子,在UBC-MNCs诱导分化后表达明显增强。脐血能够成为NSCs的新来源。
Objective To investigate the feasibility of using human umbilical cord blood mononuclear cells (UBC-MNCs) to differentiate into neural stem cells (NSCs) in vitro and to observe the expression of Nestin mRNA in NSCs. Methods UBC-MNCs were isolated from human umbilical cord blood by density gradient centrifugation. Neurobasal medium containing human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF) and B27 was induced to differentiate into NSCs The differentiation and morphological characteristics of NSCs were observed. The expression of nestin, neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry in cultured cells. The expression of Nestin mRNA in cultured cells before and after induction was detected by RT-PCR. RESULTS: UBC-MNCs could be induced to differentiate into neuron-like cells, forming typical NSC clones and expressing Nestin, NSE and GFAP-labeled antigens. The expression of Nestin, NSE and GFAP in pre-induction cells were very low. Nestin positive cells Number of peaks, and then began to decline, NSE, GFAP positive cells gradually increased. Brdu labeled target cell positive rate of 90%. The expression of Nestin mRNA was low in pre-induction cells, and gradually increased at 3d, 6d, 9d, 12d (P <0.05). CONCLUSION: UBC-MNCs-derived NSCs can be obtained by in vitro culture. Nestin gene is a key regulator of proliferation and differentiation of NSCs, which is significantly enhanced after UBC-MNCs differentiation. Cord blood can become a new source of NSCs.