C57BL/6J胚鼠听皮层神经干细胞分离培养的研究

Primary Culture of Neural Stem Cells From the Auditory Cortex of Embryonic C57BL/6J Rat

目的探讨酶消化法和机械吹打法对胚鼠听皮层神经干细胞分离培养的影响和作用。方法取孕14d的C57BL/6J小鼠12只,分成两组,每组6只,于解剖显微镜下分离胚鼠大脑听皮层区域,分别用酶消化法和机械吹打法进行原代培养。用免疫荧光技术对培养后的神经干细胞进行Nestin鉴定,并进行神经干细胞分化能力鉴定,利用CCK-8检测两种方法培养的细胞的增殖情况。结果利用酶消化法分离下来和增殖形成的神经干细胞明显多于机械吹打法,且形成的神经干细胞球比较规则,酶消化法分离下来的细胞数目为(1.57±0.05)×105个/ml,而机械吹打组分离下来的细胞数目为(1.07±0.04)×105个/ml,差异有统计学意义(P<0.05);CCK-8检测酶消化法增殖快于机械吹打法,差异有统计学意义(P<0.05)。两种方法培养的细胞免疫荧光鉴定Nestin表达均为阳性,诱导分化形成表达β微管蛋白(β-tubulinⅢ)阳性的神经元和表达胶质纤维酸性蛋白(GFAP)阳性的星形胶质细胞。结论用酶消化法对C57BL/6J胚鼠听皮层神经干细胞进行分离培养优于机械吹打法。听皮层神经干细胞具有自我增殖克隆和多向分化能力。

Objective To investigate the difference of trypsin--dispersed method and mechanical triturated methode for autaring anditory cortex neural stem ceils from embryonic C57BL/6J rat. Methods Embryonic cortex was dissected from timed--pregnant C57BL/6J rat on E14. Trypsin--dispersation and mechanical trituration were used to separate and culture neural stem cells. Fluorescence immunocytochemistry was carried out to examine the expression of NSCs marker （Nestin） and the ability of differentiation of NSCs. Then CCK-8 was used to detect cell proliferation of NSCs at different time points. Results The cell numbers isolate by trypsin--dispersation were more than these of by mechanical trituration. NSCs proliferated more quickly in trypsin--dispersation group than that in mechanical trituraton group through detection of CCK--8 （P〈0.05）. These NSCs stained Nestin could differented into neuron examined by β-tubulin Ⅲ ,astrocyte examined by glial fibrillary acidic protein（GFAP）. Conclusion The trypsin-dispersation mettiod was more convenient and faster than that of mechanical trituration. NSCs had self-- renewal ability and neural differentiation notential.