NGF联合b-FGF对胚胎大鼠隔区来源神经干细胞分化为神经元的诱导作用

摘要

目的 探讨神经生长因子(NGF)联合碱性成纤维细胞生长因子(b-FGF)诱导隔区来源神经干细胞(NSC)分化为神经元的作用.方法 体外分离培养胎鼠大脑隔区来源NSC,细胞免疫荧光检测其Nestin、GFAP、β-TubulinⅢ表达,对其分化潜能进行鉴定;将神经球分为对照组、NGF组(NGF 50 ng/mL)及NGF联合b-FGF组(NGF 50 ng/mL,并逐渐撤除bFGF)诱导分化7 d,采用细胞免疫荧光检测β-TubulinⅢ阳性细胞比例.结果(1)胚胎大鼠隔区来源神经干细胞,通过含EGF及b-FGF的无血清培养可获得大量Nestin阳性神经干细胞球,撤除丝裂原因子EGF及b-FGF后,无血清培养条件下,可自分化为GFAP阳性星形胶质细胞及β-TubulinⅢ阳性神经元;(2)对照组、NGF组(NGF 50 ng/mL)及NGF联合b-FGF组β-TubulinⅢ阳性细胞比例分别为(21.80±2.81)％、(38.04±3.86)％、(50.01±7.65)％,后两组神经元分化率均高于对照组(P＜0.05),且NGF联合b-FGF组神经元分化率高于NGF组(P＜0.05).结论 胚胎大鼠脑隔区可分离培养出Nestin阳性NSC,并可自分化为GFAP阳性星形胶质细胞及β-TubulinⅢ阳性神经元;NGF可体外诱导隔区来源神经干细胞分化为神经元,在逐渐撤除bFGF条件下,NGF诱导其分化为神经元的作用更佳.%Objective To investigate the effect of NGF combined with b-FGF on the differentiation of embryonic rat septum derived neural stem cells into neurons.Methods Embryonic rat septum derived neural stem cells were cultured in vitro. The differentiation capacity, and the expression of nestin, GFAP, β-Tubulin Ⅲ were detected by cell IF staining. The neural spheres were divided into control group, NGF group (NGF 50 ng/mL), NGF combined with b-FGF (NGF 50 ng/mL, and gradually remove the b-FGF) group. The ratio of β-Tubulin Ⅲ positive cells were detected by IF staining. Results The neural stem cells derived from embryonic rats septum can be obtained a large number of nestin positive neurospheres through serum-free culture containing EGF and b-FGF. When removing mitogenic factors EGF and b-FGF, the stem cells could be naturally differentiated into GFAP and β-Tubulin Ⅲ positive cells. In the control group, NGF group and NGF combined with b-FGF group, the ratio of β-Tubulin Ⅲ positive cells were (21.80±2.81) ％, (38.04±3.86) ％ and (50.01％±7.65) ％, respectively. The neuronal differentiation rate in the combined group was significantly higher than NGF group (P＜0.05), and they were both significantly higher than that in control group (P＜0.05). Conclusion Nestin positive NSC can be isolated and cultured from the septum of embryonic rats, these neurospheres can be naturally differentiated into GFAP and β-Tubulin Ⅲ positive cells. NGF can induce differentiation of neural stem cells from septum origin into neurons in vitro, when gradually removing the b-FGF, the differentiation rate of neurons increases.