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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。
OBJECTIVE: To investigate the morphological and functional changes of spinal cord microglia induced by glutamate and to explore whether glutamate could activate microglia and activate the optimal concentrations and time points. Methods Spinal cord microglial cells were cultured in vitro. Cell culture medium was used as a negative control group, 50μM ATP stimulated cells for 1 hour as positive control group, and glutamate was treated with different concentration (500μM, 1000μM and 2000μM) The microglia were identified by immunocytochemistry at different time points (30min, 1h and 2h), and the levels of tumor necrosis factor (TNF-α) and superoxide dismutase (TNF-α) were measured by enzyme-linked immunosorbent assay Nitric oxide (NO) expression levels. Results After incubated with 500μM glutamate for 1h, microglial cells released TNF-α and started to be activated. The release of TNF-α significantly increased at 2h (P <0.05). For 1000μM and 2000μM groups, TNF-α increased significantly at three time points (P <0.05), but not in a time-dependent and concentration-dependent manner. In the 1000μM 2h group, TNF-α was the most abundant (P <0.001). Compared with the control group, glutamate concentration of 1000μM and 2000μM and incubation time required for 2h, the release of spinal cord microglia NO increased significantly (P <0.05). Conclusion Glutamate activates microglial cells, probably 1000 μM, which is the optimal concentration for microglia activation.