新生大鼠大脑皮质神经元原代培养方法的建立及其活性鉴定

Primary culture and viability assessment of the cerebral cortical neurons from newborn SD rat

采用倒置相差显微镜、环境扫描电子显微镜对新生24h内SD大鼠大脑皮质原代培养的神经元进行了形态学观察，建立了大脑皮质神经元原代培养方法。应用2．5μg／mL阿糖胞苷（Ara-C）对培养3d的神经元进行24h干预，去除神经胶质细胞、成纤维细胞、神经干细胞等杂质细胞，以提高神经元产率；用Nissl’s染色法进行神经元鉴定；利用微量滴定（MTT）法测定了一个培养周期（约10d）的神经元活性分布。光学显微镜观察结果表明，培养至第4h绝大多数神经元已贴壁生长，胞体上可见1～2个细小突起；培养至第3d、第6d时，Nissl’s染色结果显示，加Aralc能显著提高神经元纯度（≥929／6）；MTT结合形态学观察结果表明，神经元培养至第6d时活性最强，细胞呈卵圆形、蜘蛛状、锥体状等多种形态，细胞胞体饱满，光晕明显，神经网络形成完整。

To develop a suitable primary culturing method of cortical neurons of newborn SD rats（≤24 h-old） and to establish an experiment cell model of the neurons, the morphological changes of neuron cells cultured in vitro were observed by the inverted light microscope and the scanning electron microscope. After 3 days 2.5 μg/mL of arabinoside cytarabine（Ara-C） was added to the culture for 24 h to inhibit the outgrowth of glial cells,mechanocytes and nerve stem cells, etc. Evaluation of the nerve cells was carried out by the Nissl＇s staining method. Viability of the neurons in a cell cycle（about 10 d） was investigated by the measurement of optical density（OD） values at 570 nm using MTT method. A systematic method has been established for primary cultivating of cortical neurons. Observations indicated that the cortical neurons were affixed to the culture after 4 h, and the nerve cells had 1--2 small processes, The Nissl＇s staining results show that the purity（≥92%） of the cortical neurons rose apparently on days 3 and 6 post-beginning of culturing with Ara-C inhibition. The MTT experiment show that the viability of the neurons was the highest,and they are oval, araneiform and pyramidal and formed an integrated neural network on day 6 post-beginning of culturing.