【摘要】 目的:研究钙防卫蛋白S100A9刺激体外星形胶质细胞后细胞外谷氨酸浓度变化及其对神经元影响和调控机制。方法:分离培养新生C57BL/6小鼠大脑皮层星形胶质细胞和神经元；Amplite荧光法检测星形胶质细胞培养上清谷氨酸浓度；Real time RT-PCR和Western Blot分别检测星形胶质细胞谷氨酸转运体（GLT-1） mRNA和蛋白表达；Fluo-4荧光探针法检测星形胶质细胞胞内Ca²⁺浓度；转录组测序并结合KEGG分析筛选星形胶质细胞谷氨酸浓度变化的调控机制；S100A9刺激星形胶质细胞的培养上清（CS）干预神经元后，末端脱氧核苷酸转移酶标记法（TUNEL）检测神经元凋亡，CCK-8试剂盒检测神经元存活。结果:S100A9刺激星形胶质细胞后细胞外谷氨酸浓度增加，GLT-1 mRNA和蛋白表达减少，细胞内Ca²⁺浓度增加。差异表达基因KEGG富集在核因子-κB（NF-κB）信号通路、toll样受体（TLRs）信号通路和肿瘤坏死因子（TNF）信号通路等。培养上清组神经元凋亡率高于对照组。结论:S100A9可能通过激活TLR4/NF-κB通路抑制GLT-1依赖的谷氨酸摄取和促进星形胶质细胞Ca²⁺依赖的谷氨酸释放，上调细胞外谷氨酸水平，这可能进一步导致神经元损伤。更多还原

【Abstract】 Objective: To investigate the variations of extracellular glutamate levels and its effects on neurons in response to stimulation of astrocytes with calprotectin S100A9 in vitro, and to explore these regulatory mechanisms. Methods: Neonatal C57BL/6 mouse cortical astrocytes and neurons were isolated and cultured; Amplite fluorescence assay was used to detect glutamate levels of astrocytes culture supernatant; Real time RT-PCR and Western Blot were used to measure glutamate transporter-1（GLT-1） mRNA and protein expression in astrocytes respectively. Fluo-4 fluorescent probe assay was used to measure intracellular Ca²⁺ concentration in astrocytes; Transcriptome sequencing combined with the Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway analysis to screen regulatory mechanisms of glutamate concentration changes in astrocytes; After the intervention of culture supernatant（CS） obtained from astrocytes treated with S100A9, neuronal apoptosis was detected by terminal deoxynucleotidyl transferase labeling（TUNEL） and neuronal viability was detected by CCK-8 kit. Results: Upon stimulation of astrocytes with S100A9, extracellular glutamate concentration was increased, GLT-1 mRNA and protein expression levels were reduced and intracellular Ca²⁺ concentration was increased. Differentially expressed genes were enriched in regions associated with nuclear factor-κB（NF-κB） signaling pathway, toll-like receptors（TLRs） signaling pathway and tumor necrosis factor（TNF） signaling pathways etc. The apoptosis rate of neurons in culture supernatant group was higher than that in control group. Conclusion: S100A9 probably elevate extracellular glutamate levels through activating the TLR4/NF-κB pathway to inhibit GLT-1 dependent glutamate uptake and promote astrocytes Ca²⁺ dependent glutamate release, which may further contribute to neuronal injury.更多还原