中文摘要：

在急性、慢性神经退行性疾病和炎症引发的神经系统疾病的发病机制中，兴奋性毒性可能是造成后期神经元死亡的共同途径．小脑颗粒神经元谷氨酸兴奋性毒性模型是研究上述过程的重要实验手段，该模型的稳定性和可重复性是开展相关研究的重要基础．然而，文献报道的建模方法条件各异，说法不一，很难适从．本工作针对小脑颗粒神经元谷氨酸兴奋性毒性模型建立的关键环节，包括小脑颗粒神经元的培养、兴奋性毒性刺激条件的确定，毒性标志性指标的表征，分别进行了比较和优化，从培养皿的包被、神经元消化、兴奋性刺激的溶液介质选择、神经元刺激的最佳时间及谷氨酸的最佳刺激浓度等方面分别给出了优化条件．通过特征性钙离子曲线、NMDA受体特异性抑制剂MK-801的干预作用以及c-fos。基因转录水平的动力学变化等指标，确认了毒性模型的成功建立．本工作不仅对建立小脑颗粒神经元谷氨酸兴奋性毒性模型的实验室具有重要参考意义，而且，其针对不同条件分析比较的结果及优化原则，对其他神经毒性模型的建立也具有普遍参考意义．

英文摘要：

Glutamate excitotoxicity of cerebellar granule neurons （CGN） is often used as a model for studying the pathogenesis of acute, chronic neurodegenerative diseases and inflammation-induced neurological diseases. However, reports of culture methods for CGN and conditions for their stimulation vary considerably, making it difficult to compare reports from different laboratories. Here, we compare different methods and optimize the model as follows： Culture plates were coated overnight at room temperature with Poly-L-lysine at a concentration of over 50 mg/L. Cerebellum tissues were then shredded and digested with trypsin （0.025%） for 15 min at 37 ℃ with shaking in the presence of DNase I to eliminate DNA released from broken cells. In order to remove impurities and cell debris, samples were centrifuged respectively after tissue shredding, digestion and mechanical dispersion. Samples were then homogenized by pipetting and allowed to settle three times to increase the yield of CGN. Best results were achieved when settling time was extended to 15 min before collecting the supernatant cells. Conditional BME was used as the culture medium since our data showed that CGNs are more sensitive to glutamate in conditioned than in flesh BME, neurobasal medium or Locke＇s buffer. Cells cultured for 9 DIV （days in vitro） were treated with 100 μmol/L glutamate to generate moderate excitotoxicity. The model was validated by examining intracellular calcium dynamics and c-fos expression. Results show that this method is stable and reproducible and may be helpful for researchers using the excitotoxicity model to study neuropathological processes.