缺氧对鼠视网膜Müller细胞GLAST和GS表达及功能的影响

Effect of Hypoxia on The Glutamate Transporter and Glutamine Synthetase in Mouse Retinal Müller Cells

研究了缺氧对鼠视网膜Müller细胞谷氨酸转运体(L-glutamate/L-aspartate transporter,GLAST)和谷氨酰胺合成酶(glutamine synthetase,GS)表达的影响,及对谷氨酸摄取的作用.采用出生3～7天的小鼠视网膜组织进行Müller细胞培养,采用125μmol/L的氯化钴(CoCl2)溶液分别进行缺氧干预6、12、24、48和72 h,不加CoCl2溶液培养的Müller细胞为正常对照.采用RT-PCR法、Western blot法和免疫细胞化学染色法检测GLAST和GS的表达,并检测谷氨酸摄取及细胞凋亡情况.结果显示,缺氧早期GLAST表达较正常对照组增强(P<0.001),CoCl2溶液干预12 h后达到最强(P<0.05),之后逐渐降低.CoCl2溶液干预72 h后GLAST表达与正常对照组相比无明显差异(P>0.05).而缺氧也使GS的表达较正常对照组增加(P<0.001),CoCl2溶液干预48 h后GS表达最强(P<0.001),之后开始下降.缺氧促进Müller细胞对谷氨酸的摄取,CoCl2溶液干预48 h后L-[3,4-3H]-谷氨酸的摄取量最大(P<0.005),之后开始下降.CoCl2溶液干预后,Müller细胞死亡数较正常对照组无明显差异(P>0.05).结果表明,在一定时间范围内缺氧能够增强Müller细胞GLAST及GS的表达,增加谷氨酸的摄取.但持续缺氧最终会引起Müller细胞功能失代偿,从而导致谷氨酸的代谢能力降低.

The effect of hypoxia on expression and function of L-glutamate/L-aspartate transporter （GLAST） and glutamine synthetase（GS） was investigated in mouse retinal Muller cells（RMCs）. Mouse RMCs were cultured by enzymatic digestion method. RMCs cultures were treated with CoCl2 （125 μmol/L） for 6 h, 12 h, 24 h, 48 h or 72 h respectively in vitro. RMCs cultures were maintained without CoCl2 in normal control group. The expression of GLAST and GS was determined by RT-PCR, Western blotting and immunocytochemical staining. L-[3,4-^3H]- glutamic acid uptake was used to quantify glutamate uptake function of RMCs. The apoptosis of RMCs was confirmed by annexin V-FITC/PI staining. In early-stage of CoCl2-induced hypoxia （treated with CoCl2 for 6 h, 12 h, 24 h or 48 h）, the expression of GLAST was up-regulated （P 〈 0.001） and reached the maximum when RMCs have been treated with CoCl2 for 12 h （P 〈 0.05）. After RMCs having been treated with CoCl2 for 72 h, the expression of GLAST had no difference compared to normal control （P 〉 0.05）. CoCl2-induced hypoxia （treated with CoCl2 for 6 h, 12 h, 24 h, 48 h or 72 h） also up-regulated the expression of GS （P〈 0.001） which reached the maximum when RMCs have been treated with CoCl2 for 48h （P 〈 0.001）. The L-[3,4-^3H]-glutamic acid uptake of hypoxia groups were the higher than normal control group （P 〈 0.05）, and after having treated RMCs with CoCl2 for 48 h, the L-[3,4-^3H]-glutamic acid uptake was the highest （P 〈 0.005）. Treatments with CoCl2 did not induce apoptosis in RMCs. In early hypoxia stage, GLAST and GS were up-regulated and extracellular glutamate uptake increased. However, continued hypoxia causes gradual dysfunction and reduction of GLAST and GS, as well as glutamate uptake.