新生大鼠海马神经元的原代培养与鉴定

Primary Culture and Identification of Hippocampal Neuron from Rats

目的：探讨建立简便高效的大鼠海马神经元原代培养的纯化方法。方法：（1）离体培养新生24h内SD乳鼠海马神经元，每天在倒置相差显微镜下进行神经元的形态学观察；（2）MTY法检测培养不同时间段神经元的活力变化，并绘制生长曲线；（3）纯化培养9d神经元利用免疫荧光染色法检测β-微管蛋白Ⅲ（β-tublinⅢ）的表达率。结果：（1）倒置相差显微镜下观察原代培养的海马神经元，刚分离种植时可见神经细胞为圆形，24h大多数细胞贴壁，3d细胞突起增大、增粗，培养5～7d神经元胞体丰满，突起交织成的网络更密集，培养14—16d后，神经元开始退化；（2）生长曲线结果发现海马神经元体外培养经历了4个时期，即生长潜伏期、对数生长期、平台期、生长停滞期；（3）经p—tublinUI鉴定海马神经元纯度为（93．0％±2．5％）。结论：本实验方法步骤简便，可获得纯度较高的海马神经元，为研究与海马神经元相关的疾病提供了实验基础。

Objective： To establish an efficient method of primary culture of hippocampal neurons from rats. Methods： Hippocampal regions of new - born SD rats （ 〈 24h） were harvested. The morphological changes of the cultured neurons were observed under a light invert microscope. The growth curve of hippocampal neurons was determined by MTr. Double immunofluorescence staining of β -Tubulin and karyon was applied to assess the culture purity. Results： The newly inoculated hippocampal neurons were round, small. Most of neurons adhered to the dishes after 24 hours culture. On the 3rd day, most of the cell morphologies were fusiform, triangular, with different lengths of slender protrusions. With time extending, cell body enlarged , furthermore, prominence enlarged and elongate, and then connected with each other. After cultivated for 14d, cells began period, exponential degenerate. Primary cultivated hippocampal neuron showed a curve with latency growth phase, growth plateau phase, and stasis period. The cells grew well and the cell purity was demonstrated to be satisfactory （93.0%± 2.5% ）. Conclusion： The established cultural method is a convenient with high activity and purity, which provides a basis for studing the related disorders of the hippocampal neurons.