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目的观察高浓度谷氨酸刺激条件下Muller细胞的变化以及促红细胞生成素(erythropoietin,EPO)的保护作用是否与维持Muller细胞的谷氨酸转运功能相关。方法用MTT法检测不同浓度谷氨酸处理大鼠原代视网膜Muller细胞和大鼠视网膜Muller细胞系rMC-1所引起的细胞活力变化。选择能显著降低细胞活力的最低谷氨酸剂量建立细胞损伤模型,并研究不同浓度EPO干预后细胞活力的变化。TUNEL(terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)法检测各组细胞凋亡情况,Western印迹法检测谷氨酸转运体GLSAT在蛋白水平的变化。结果无论是在原代大鼠Muller细胞还是rMC-1细胞系中,谷氨酸的细胞毒性作用均呈剂量依赖趋势。原代Muller细胞中,12 mmol/L谷氨酸使细胞活力降低15.5%,10 mmol/L谷氨酸使rMC-1细胞活力降低15.6%;此时细胞凋亡明显增加,GLAST表达降低。0.2 U/ml和0.5 U/ml EPO分别对原代Muller细胞和rMC-1细胞的保护作用最佳,细胞凋亡数量显著减少,并防止了GLAST蛋白水平的降低。结论体外高浓度谷氨酸可损伤Muller细胞,EPO可以通过维持谷氨酸转运体GLAST水平等机制维持Muller对谷氨酸的正常摄取、抑制凋亡发生。
Objective To observe the changes of Muller cells and the protective effect of erythropoietin (EPO) under high concentration of glutamate on the maintenance of glutamate transport function in Muller cells. Methods MTT assay was used to detect the changes of cell viability induced by different concentrations of glutamate in rat primary retinal Muller cells and rat retinal Muller cell line rMC-1. Select the lowest glutamate dose can significantly reduce cell viability to establish a cell injury model, and study the changes of cell viability after different concentrations of EPO intervention. Cell apoptosis was detected by TUNEL assay, and Western blotting was used to detect the change of glutamate transporter GLSAT at the protein level. Results The cytotoxic effect of glutamate showed a dose-dependent manner both in primary rat Muller cells and rMC-1 cell lines. In the primary Muller cells, 12 mmol / L glutamate decreased cell viability by 15.5%, and 10 mmol / L glutamate decreased the viability of rMC-1 cells by 15.6%. At this time, apoptosis increased and GLAST expression decreased. The protective effects of 0.2 U / ml and 0.5 U / ml EPO on primary Muller cells and rMC-1 cells were the best, the number of apoptotic cells was significantly reduced, and the decrease of GLAST protein level was prevented. Conclusion High concentration of glutamate in vitro can damage Muller cells. EPO can maintain the normal uptake of glutamate and inhibit the apoptosis by maintaining GLAST level of glutamate transporter.