关于谷氨酸激活脊髓小胶质细胞的研究

目的通过观察谷氨酸对脊髓小胶质细胞在形态和功能方面的改变,探讨谷氨酸能否激活脊髓小胶质细胞以及激活的最佳浓度和时间点。方法原代离体培养脊髓小胶质细胞,分别用细胞培养液作为阴性对照组、50μM的ATP刺激细胞1小时作为阳性对照组以及谷氨酸通过不同浓度（500μM,1000μM和2000μM）分别作用三个不同的时间点（30min、1h和2h）作为实验组,通过免疫细胞化学方法鉴定小胶质细胞,通过酶联免疫吸附法（ELISA）测定上清液中肿瘤坏死因子（TNF-α）和一氧化氮（NO）的表达水平。结果 500μM的谷氨酸孵育1h,小胶质细胞释放TNF-α并开始被使激活,在2h TNF-α释放量大大增加（P〈0.05）。对于1000μM和2000μM组,TNF-α在三个时间点增加量都非常明显（P〈0.05）,但并不呈现时间依赖性和浓度依赖性趋势。在1000μM 2h组,TNF-α释放量最多（P〈0.001）。而与对照组相比,谷氨酸浓度达到1000μM和2000μM且孵育时间需持续2h,脊髓小胶质细胞释放的NO才会明显增加（P〈0.05）。结论谷氨酸能够激活小胶质细胞,可能1000μM是其激活小胶质细胞的最佳浓度。

Spinal Microglia can be Activated by Glutamate

Objective We aimed to explore whether glutamate could activate spinal microglia by observing the changes of morphology and function of spinal microglia and to find out the best stimulating concentration of glutamate and best time point.Methods Primary culture spinal microglia,the culture medium as negative control group,ATP 50μM 1h as positive control group and glutamate at different concentration（500μM,1000μM and 2000μM） and different time points（30min,1h and 2h） stimulated spinal microglia as experimental groups.Immunocytochemistry was used to identify microglia and ELISA assay was used to test the level of TNF-α and NO.Results When 500μM glutamate was administrated for 1 hour,the microglia started to be activated by releasing TNF-α and increased dramatically for 2 hours（P0.05）.For group with 1000μM and 2000μM,the increase of TNF-α are all significantly in three time points（P0.05）.But it was not appeared to be time-dependent and dose-dependent trend.Group with 1000μM glutamate for 2 hours reached peak value（P0.05）.While compared with control group,only when concentration of glutamate reached 1000μM and 2000μM and time last for 2 hours,can spinal microglia increase NO release significantly（P0.05）.Conclusion Glutamate could activate spinal microglia,maybe 1000μM is the most effective concentration for glutamate to make microglia active.