FAC对体外培养皮质神经干细胞分化率的影响

EFFECTS OF FERRIC AMMONIUM CITRATE ON DIFFERENTIATION OF CORTEX NEURAL STEM CELLS IN VITRO

目的探讨不同浓度柠檬酸铁铵(FAC)对体外培养的大鼠皮质神经干细胞(NSCs)细胞活力和分化率影响。方法体外培养胚胎14~15d的皮质NSCs;应用CCK-8细胞增殖和毒性检测试剂盒检测经FAC处理后NSCs细胞活力的改变,免疫荧光染色检测Nestin、β-tubulinⅢ和胶质纤维酸性蛋白(GFAP)阳性细胞,Western Blot检测NSCs的β-tubulinⅢ和GFAP蛋白表达水平。结果分离培养的皮质NSCs能增殖形成神经球,且经分化培养基诱导后可分化为β-tubulinⅢ或GFAP阳性的细胞。不同浓度(10~100 mg/L)的FAC均可降低细胞活力(F=55.86,q=9.154~15.880,P<0.01)。不同浓度的FAC不影响NSCs的β-tubulinⅢ和GFAP蛋白表达水平(P>0.05)。结论 FAC可降低皮质NSCs细胞活力,但不影响NSCs向神经元和星形胶质细胞的分化比例。

Objective To investigate the effects of ferric ammonium citrate（FAC）on the viability and differentiation of rat cortex neural stem cells（NSCs）in vitro. Methods Cortex NSCs were isolated from the cerebral cortex of rat embryos cultured in vitro on embryonic days 14-15.The effect of FAC on the viability of cortex NSCs was determined by Cell Counting Kit-8 assay.Immunofluorescence was used to identify Nestin-,β-tubulin Ⅲ-,and glial fibrillary acidic protein（GFAP）-positive cells.The expression levels ofβ-tubulinⅢand GFAP proteins in NSCs were assessed by Western blot. Results Cortex NSCs cultured in vitro could proliferate into neural spheres and differentiate intoβ-tubulin Ⅲ-or GFAP-positive cells.FAC significantly inhibited the viability of NSCs at concentrations ranging from 10 to 100 mg/L（F=55.86,q=9.154-15.880,P〈0.01）.Different concentrations of FAC had no effects on the expression levels ofβ-tubulinⅢ or GFAP proteins in NSCs（P〉0.05）. Conclusion FAC reduces cell viability but has no effect on the differentiation of cortex NSCs into neurons or astrocytes.