摘要
目的探讨谷氨酸对大鼠视网膜Mller细胞超微结构和谷氨酰胺合成酶(GS)表达的影响。方法取5～7d清洁级Wistar乳鼠视网膜,在含质量分数10%新生牛血清的Dulbecco改良Eagle培养基中传代培养大鼠视网膜Mller细胞。第3代Mller细胞培养基中加入终浓度分别为10、20、50、100、200μmol/L的谷氨酸作用10min。正常对照组Mller细胞培养基中未加入谷氨酸。通过免疫细胞化学法检测GS的表达鉴定视网膜Mller细胞。透射电镜观察各组Mller细胞超微结构的改变。采用Westernblot法半定量检测GS的表达。结果培养的Mller细胞中,GS阳性细胞达95%以上。透射电镜下正常对照组Mller细胞超微结构正常,谷氨酸10、20、50μmol/L组Mller细胞超微结构无明显改变。100μmol/L谷氨酸组可见Mller细胞线粒体肿胀和染色质的凝集、边聚。在200μmol/L谷氨酸组可见Mller细胞空泡样变性和凋亡小体。Westernblot结果显示,10、20、50μmol/L组GS在Mller细胞中表达较正常对照组升高,差异均有统计学意义(q=29.32、q=75.54、q=48.36、q=130.20,P<0.01),50μmol/L谷氨酸组GS的表达达高峰,100μmol/L谷氨酸组表达开始下降,200μmol/L谷氨酸组的表达低于正常对照组(q=46.67,P<0.01)。结论谷氨酸浓度影响视网膜Mller细胞中GS的表达,谷氨酸浓度超过50μmol/L可引起视网膜Mller细胞超微结构的改变。
Background Glutamic acid is an important neurotransmitter of retina.Excessive accumulation will induce neurotoxicity to nervous tissue.A lot of researches of the effect of glutamic acid on retina tissue focus on retinal nerve cells,but its effect on retinal Müller cells is still seldom.Objective Present study was to investigate the effects of glutamic acid on ultrastructure of retinal Müller cells and the expression of glutamine synthetase in cultured retinal Müller cells.Methods Retinal Müller cells were isolated from the eye of 5-to 7-day-old clean Wister rats and cultured in dulbecco’s modification of eagle’s medium dulbecco with 10% calf serum.10,20,50,100,200 μmol/L L-glutamic acid was used in the medium of the third generation of cells for 10 minutes.No L-glutamic acid was used in the normal control group.Cultured Müller cells were identified by immunochemistry to detect the expression of glutamine synthetase.The changes of the ultrastructure of cultured Müller cells were examined under the transmission electron microscope.The expression of glutamine synthetase was semi-quantitatively detected using Western blot.Results Cultured cells showed the consistent shape with the positive reaction for glutamine synthetase in over 95% cells.The Müller cells in normal control group presented the normal cellular organs and ultrastructure.In 10,20,50 μmol/L glumatic acid groups,no obvious change of ultrastructure was found in Müller cells under the transmission electron microscope.Swelling of mitochondrion,agglutination and margination of chromatic in the Müller cells were exhibited in 100 μmol/L glutamic acid group,and vacuoles apoptosis body in the Müller cells could been seen in 200 μmol/L glutamic acid group.Western blot assay revealed that glutamine synthetase was positively expressed in 10,20,50 μmol/L glutamic acid groups and the expression reached the peak at 50 μmol/L glutamic acid groups,and then the expression level of glutamine synthetase declined in 100 and 200 μmol/L glutamic acid groups.The expression levels of glutamine synthetase was significantly higher in 10 μmol/L,20 μmol/L,50 μmol/L,100 μmol/L group glutamic acid group compared with control group （q=29.32,q=75.54,q=48.36,q=130.20,P0.01）,and that in 200 μmol/L glutamic acid group was lower than control group（q=46.67,P0.01）.Conclusion The dynamic change of expression of glutamine synthetase in Müller cells is related to concentration of glutamic acid.Over 50 μmol/L of glutamic acid causes the damage of ultrastructure of Müller cells.
出处
《眼科研究》
CSCD
北大核心
2010年第10期933-936,共4页
Chinese Ophthalmic Research
