摘要
目的:建立体外谷氨酸诱导神经元兴奋损伤模型,探索其凋亡发生是否通过线粒体信号转导途径介导的细胞色素C(CytC)释放而实现,为今后干预性使用神经保护剂提供依据。方法:分离及培养新生Wistar大鼠海马神经元,选用合适谷氨酸浓度建立神经元损伤模型;利用LDH测定及流式细胞仪Annexin V/PI双染色法检测谷氨酸暴露后不同时点神经元凋亡及坏死的动态改变;采用Western blotting法检测caspase-3活性及线粒体内和胞浆内CytC水平动态变化。结果:谷氨酸诱导神经元损伤呈明显浓度及时间依赖性,50μmol/L浓度可使LDH释放量明显增加(18.4%,P<0.05),暴露后6h凋亡率显著增加;凋亡发生前,神经元caspase-3活性已明显增高(3h),6h达高峰;线粒体CytC释放发生在caspase-3增高前,30min时胞浆内CytC水平即明显增加(P<0.05),3h胞浆内CytC水平超过线粒体内,而线粒体内CytC水平进行性减少。结论:50μmol/L谷氨酸可诱导海马神经元凋亡,凋亡机制可能是通过损伤线粒体膜,使CytC易位释放入胞浆激活caspase级联反应而致。
AIM： To set up a glutamate - induced cell damage model in cultured hippocampal neurons, and to determine whether glutamate - induced neuronal apoptosis changes and whether this process is mediated by mitochondrial signal transduction pathways involving the release of cytochrome C. METHODS ： Hippocampal neurons, isolated and cultured from new born Wistar rats, were exposed to various concentrations of glutamate. Extent of cell death was assessed by measuring the release of lactate dehydrogenase （LDH） in the culture media. Based on these data, an appropriate concentration of glutamate was selected, and all subsequent experiments were carried out under the concentration. Kinetics of glutamate -induced both apoptotic and necrotic cell death after exposure to glutamate for various times （3 -24 h） were determined by flow cytometry and LDH release. The caspase -3 protein levels and cytochrome C release from mitochondria into cytosol in hippocampal neurons were determined by Western blotting. RESULTS： Glutamate treatment induced hippocampal neurons death in dose - dependent and time - dependent manners. A significant increase in LDH release （ 18.4% ） was induced in the cells treated with 50 μmol/L glutamate, compared to control untreated cells （ P 〈 0. 05 ）. A significant increases in LDH release and apoptosis were observed at 6 h after glutamate treatment （P 〈 0. 05）. The significant increase in the level of caspase - 3 protein occurred at 3 h after glutamate exposure, which preceded neuronal cell apoptosis, and reached maximum levels at 6 h（62. 4% ）. Treatment with glutamate induced a rapid release of cytochrome C into cytosol. Cytosolic cytochrome C showed a significant increase （ P 〈 0. 05） as early as 30 min after glutamate treatment, which preceded the increase in caspase - 3 level, and after 3 h, the level of cytochrome C was higher in the cytosol compared to the mitochondria. Concomitant with these changes in cytosol, the mitoehondrial levels of cytochrome C decreased significantly. CONCLUSION： Exposure to 50 μmol/L glutamate induces apoptosis in cultured hippocampal neurons. The glutamate - induced apoptosis may be via the damage of mitochondria that results in eytochrome C release into cytosol, which activates caspase cascade and induces apoptotic cell death.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第11期2162-2166,共5页
Chinese Journal of Pathophysiology
基金
贵州省优秀人才省长资金资助项目(No.200407-4)
