新生大鼠皮层神经元体外无血清原代培养

Establishing a serum-free primary culture method for cortical neurons of new-born rats

目的探讨建立较为简便的新生大鼠皮层神经元细胞体外无血清原代培养方法。方法取新生大鼠(24h)大脑皮层组织,消化后种植在有多聚-L-赖氨酸包被的六孔培养板中,以含10%胎牛血清DEME-HG培养液种植,4～8h后换用含B27的Neurobasal培养基维持饲养。于不同时间在倒置相差显微镜下观察形态变化,分别采用RT-PCR、Western blot、免疫组织化学对神经元特异性标记物神经元特异性烯醇化酶(NSE)基因及蛋白进行鉴定。结果 2～8h神经元细胞贴壁,随着时间延长,形态多变,突起逐渐增多,神经元突起间相互接触形成网络,培养7～10d神经元胞体最为丰满,通过RT-PCR、Western blot和免疫组织化学证明所分离培养的是神经元细胞。结论该方法简单易行,神经元纯度较高,可作为神经元体外培养的良好模型用于以后的研究。

Objective To establish a serum-free primary culture method for cortical neurons of new-born rats .Methods The cortical tissue was digested and the cells were planted in the medium containing 10% fetal bovine serum ,and then maintained feed-ing with neurobasal medium containing B27 after 4 to 8 h .The morphology was observed under phase-contrast microscope .RT-PCR ,Western blot and immunocytochemistry were applied to identify the expression of NSE gene and protein in neurons .Results A large number of neurons began to adhere to the cover glasses after 2 to 8 h .They showed different shapes-shuttle ,triangle pyram-idal ,or no regular after clinging to the plate .Their processes connected to nets and were different in length and thickness .They well developed at the 7th to 10th day .The isolated and cultured cells were confirmed as neurons by RT-PCR ,Western blot and immuno-cytochemistry .Conclusion This technique is an easy and practical tool for primary culture of new-born rats cortical neurons with high purity ,and can be used as an in-vitro model of research .