米诺环素在L-谷氨酸诱导的视网膜神经节细胞损伤中的保护作用及分子机制  

Protective effect of minocycline on glutamate-induced retinal ganglion cell injury and molecular mechanism

作　　者：刘煜[1] 唐罗生[1] 

机构地区：[1]中南大学湘雅二医院眼科,长沙410011

出　　处：《中华眼科杂志》2010年第9期802-809,共8页Chinese Journal of Ophthalmology

摘　　要：目的探讨米诺环素在L-谷氨酸诱导的视网膜神经节细胞（RGC）毒性中的保护作用和分子机制。方法实验研究。原代小鼠RGCs体外培养24h后，随机分为3组：对照组，L-谷氨酸组（100μmol／L、500μmol／L、1mmol／L和2mmol／L）及L-谷氨酸＋米诺环素组（30μmol），观察不同浓度L-谷氨酸对RGC的存活率与轴突生长的损伤作用及米诺环素的保护作用。体内实验，将雌性B6小鼠随机分为实验组（30只）和对照组（30只）。两组小鼠腹腔内分别注射米诺环素（实验组，60mg／kg）或生理盐水（对照组），每天1次，连续7d。第2天时，两组小鼠玻璃体腔内注射2μl L-谷氨酸（2mmol／L），诱导RGC损伤。免疫组化染色分析β-Ⅲ-tubulin阳性细胞数目变化及视网膜神经胶质酸性蛋白（GFAP）表达情况，Real-time PCR和免疫印迹法分别检测小鼠视网膜组织中干扰素1（IFN-1）、白细胞介素（IL-1）、肿瘤坏死因子α（TNF-α）、GFAP与波形蛋白（Vimentin）的mRNA及蛋白表达水平。结果体外实验显示，与对照组相比，L-谷氨酸降低RGC的存活率，与剂量和干预时间呈负相关。同时L-谷氨酸可明显抑制RGC轴突的生长，RGC轴突长度〉2BL、1～2BL、〈1BL占总细胞数比例分别从50．38％、7．83％和3．72％降至31．43％、5．05％和1．29％。而米诺环素能明显减轻L-谷氨酸对RGC的毒性作用，改善RGC轴突生长，各组细胞比例回升至51．oo％、8．10％和2．43％，谷氨酸与对照组相比、米诺环素组与谷氨酸相比，差异有统计学意义（F=18．87，P〈0．01）。体内实验结果显示，与对照组相比，L-谷氨酸组小鼠RGC数目显著减少（45．00±10．21和68．50±2．86），而米诺环素治疗后可明显改善L-谷氨酸诱导的RGC损伤，RGC数目恢复至62．00±11．65，（F=7．6，P〈0．01）。谷氨酸处理后视网膜组织中GFAP的表达水平明显增高，而米诺环素Objective To investigate the protective effect and molecular mechanism of minocycline on toxicity of retinal ganglion cells induced by L-glutamate. Methods Primary mouse retinal ganglion cells （RGC） were isolated from mouse retinal in vitro. RGC were divided into control group, L-glutamate group, and L-glutamate ＋ minecycline group, the cell survival rate and nerve axon growth length were observed. In vivo study, B6 mice was intravitreal injected with 2 μl L-glutamate （2 retool/L） to construct a toxic damage to retinal ganglion cells animal model. One day before the beginning of the experiment, mice were daily intraperitoneal injected with minocycline （60 mg/kg, saline injected in the control group） till day 7, 133 tubulin positive ceils and retinal GFAP protein expression were evaluated by tissue i mmol/L unofluorescence assay. Real-time PCR and Western blot assay were used to detect IFN-γ, IL-1, TNF-α and GFAP and vimentin mRNA and protein expression level in retinal tissues. Results Compared with control group, RGC survival rate in L-glutamate group was significantly reduced with a dose-and time-dependent. In addition, axon growth was inhibited with the treatment of glutamic acid, while these effects were abolished in the minocycline group （F = 18. 87, P 〈 0. 01 ）. Animal study showed that the number of RGC dramatically decreased, however, expression of GFAP in retinal tissue significantly increased in L-glutamate treated mice, compared to control （F = 7. 6, P 〈 0. 01 ）. Minocycline treatment significantly improved L-glutamate-induced ganglion cell damage and significantly reduced their GFAP expression. Both mRNA and protein expression levels of IFN-γ, IL-1, TNF-α and GFAP and Vimentin in retinal tissue of L-glutamic acid group significantly upregulated compared to control, while the minocycline significantly reduced the expression of these factors. Conclusions L-glutamic acid can induce retinal ganglion cells damage, inhibit axon growth and increase inflammatory and glial-cell r

关 键 词：视网膜神经节细胞 细胞死亡 细胞保护 谷氨酸 米诺环素 

分 类 号：R774.1[医药卫生—眼科]