皮质神经元B27原代培养及MAP2鉴定

目的对原代新生鼠皮质神经元原代培养方法进行改进,以获得纯度更高、体外更易存活的神经元进行相关实验研究。方法采用胰酶消化和机械分离相结合的方法制备皮质神经元悬液,进行培养,用倒置相差显微镜观察细胞形态,利用神经元特异性标志物微管相关蛋白2(MAP2)鉴定培养神经元的纯度。结果神经元细胞存活率高,在体外培养条件下细胞状态良好,生长旺盛。培养第4天,可见多数细胞长出1、2个突起并交织成网。培养第8天细胞胞体较大,能形成典型的神经细胞网络。培养第10天,鉴定神经元纯度质量分数达90%以上。结论该方法简便可行,是体外培养神经元较理想的方法。

B27 primary culture of neonatal rat cortical neurons and MAP2 identification

Objective To improve some former culture methods of primary rat cortical neurons and get pure and long living cells for in vitro study. Methods Trypsin digestion and mechanical dissociation were adopted to conduct the culture. Morphological changes of neuron cells were observed under an inversion phase contrast microscope. Immunostaining of microtubule associated protein 2 (MAP2) was applied to assess cell purity of the culture. Results Viability of the conducted cells was higher than before. Cells grew well under the given condition. Most cells had one or two ecptoms grown and their interlacing looked like a net on the fourth day after planting. After the neurons grew successively for 8 d, the cell body became bigger and the prominences were thicker and there were more branches than that at the initial stage. The prominences of neurons interlaced very well even to form a typical nerve fiber net. On the tenth day, purity of the cultured neurons was over 90% by immunohistochemical identification. Conclusions Method introduced in the paper is a simple technique that can be used for in vitro primary culture of rat cortical neurons.