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和分子机制

发布时间：2018-08-11 18:21

【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损伤中的保护作用和分子机制。方法：体外实验,原代小鼠视网膜神经节细胞体外稳定培养24h后,分为三组：对照组、L-谷氨酸组及L-谷氨酸+米诺环素组,相应干预48h后分别观察各组间神经节细胞的存活率及神经轴突生长的差异。体内实验,将C57BL／6小鼠分为三组：对照组、L-谷氨酸组及L-谷氨酸+米诺环素组。前两组小鼠腹腔内注射生理盐水(对照组,L-谷氨酸组),第三组腹腔内注射米诺环素(L-谷氨酸+米诺环素组,60mg/kg),每天一次,连续7天,第二天时,第一组玻璃体腔内注射生理盐水,后两组小鼠玻璃体腔内注射2ulL-谷氨酸(2mM),诱导视网膜神经节细胞损伤。第八天采用组织免疫荧光法技术和激光共聚焦显微镜观察β3tubulin阳性细胞数及视网膜GFAP蛋白表达以评估视网膜神经节细胞的损伤情况。Real-time PCR和Western blot法分别检测视网膜组织中IFN-γ, IL-1, TNF-α与GFAP与Vimentin的mRNA及蛋白表达水平以初步探讨损伤的分子机制。结果：体外实验结果显示：与对照组比较,L-谷氨酸组视网膜神经节细胞的存活率明显降低,呈剂量和时间依赖性；神经轴突生长受抑制。米诺环素干预后可明显减轻L-谷氨酸导致的上述效应(P0.01)。动物实验结果显示,L-谷氨酸组小鼠神网膜神经节细胞数目较对照组小鼠显著减少,视网膜组织中GFAP的表达量明显增加(P0.01)。米诺环素治疗后可明显改善L-谷氨酸诱导的神经节细胞损伤并明显降低其GFAP的表达(p0.01)。视网膜组织中IFN-γ、IL-1、NF-α与GFAP与Vimentin的mRNA及蛋白水平在L-谷氨酸组较对照组表达明显增高,而米诺环素可显著降低这些因子的表达。结论：L-谷氨酸可诱导视网膜神经节细胞损伤、抑制神经轴突生长和上调炎症因子基因与蛋白的表达,而米诺环素具有明显改善L-谷氨酸所导致的视网膜神经细胞损伤的作用。
[Abstract]:Aim: to investigate the protective effect and molecular mechanism of minocycline on L-glutamate induced retinal ganglion cell injury. Methods: the primary mouse retinal ganglion cells were cultured in vitro for 24 hours, and were divided into three groups: control group, L-glutamic acid group and L-glutamate minocycline group. The survival rate of ganglion cells and the growth of axons were observed after 48 h intervention. In vivo, C57BL/6 mice were divided into three groups: control group, L-glutamic acid group and L-glutamate minocycline group. The first two groups were intraperitoneally injected with normal saline (control group, L- glutamic acid group), and the third group with minocycline (L-glutamate minocycline group, 60 mg / kg), once a day for 7 days, the second day, the first group was injected with normal saline in vitreous cavity. In the latter two groups, the retinal ganglion cells were injured by intravitreal injection of 2 ulL-glutamic acid (2mM). On the 8th day, the number of 尾 3tubulin positive cells and the expression of retinal GFAP protein were observed by tissue immunofluorescence technique and laser confocal microscope to evaluate the damage of retinal ganglion cells. Real-time PCR and Western blot were used to detect the retinal group. The expression levels of mRNA and protein of IFN- 纬, IL-1, TNF- 伪 and GFAP and Vimentin were studied to explore the molecular mechanism of injury. Results: compared with the control group, the survival rate of retinal ganglion cells in L-glutamic acid group was significantly decreased in a dose-and time-dependent manner, and the growth of axons was inhibited. Minocycline can attenuate the above effects induced by L-glutamic acid (P0.01). The results of animal experiments showed that the number of retinal ganglion cells in the L- glutamic acid group was significantly lower than that in the control group, and the expression of GFAP in the retina was significantly increased (P0.01). Minocycline could significantly improve the injury of ganglion cells induced by L-glutamate and decrease the expression of GFAP (p0.01). The levels of mRNA and protein of IFN- 纬 IL-1NF- 伪, GFAP and Vimentin were significantly higher in L- glutamate group than in control group, but minocycline could significantly decrease the expression of these factors. ConclusionWow-L-glutamic acid can induce retinal ganglion cell injury, inhibit axon growth and up-regulate the expression of inflammatory factor gene and protein. Minocycline can obviously improve the damage of retinal nerve cells induced by L-glutamate.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R774

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