谷氨酸转运体GLAST在小鼠视网膜兴奋性损伤后的表达

发布时间：2018-02-24 20:20

本文关键词： 谷氨酸谷氨酸转运体 GLAST 视神经保护神经节细胞　出处：《青岛大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：本实验在NMDA(N-甲基-D天门冬氨酸)所致小鼠视网膜兴奋性损伤中,观察谷氨酸转运体GLAST的蛋白表达量和mRNA随时间表达量的变化,探讨GLAST对神经节细胞凋亡的作用。方法：本实验采用健康清洁级4-8周龄C57BL/6J小鼠共54只,雌雄各半,体重约20-30g,随机分成A、B、C三组,A组6只为正常对照组,B、C为实验组各24只,其中B组为NMDA兴奋性损伤组,C组为空白对照组。B组、C组于造模后6h、24h、3d和7d四个时间点处死。处死前对每组小鼠进行暗适应,至少暗适应6小时,然后进行视网膜电生理(ERG)检查。ERG操作完毕处死动物,取每只小鼠左眼眼球做病理切片,进行HE染色、免疫组织化学及TUNEL凋亡细胞染色,右眼眼球取完整视网膜用Taqman荧光定量RT-PCR法检测视网膜中谷氨酸转运体GLASTmRNA随时间表达变化,以及与正常组比较表达差异。结果1、HE染色：(1) NMDA兴奋性损伤组：造模后6h,与正常对照组比较未见明显差异,造模后24h,视网膜的神经节细胞层开始出现凋亡形态细胞,3d凋亡细胞增多并且神经节细胞数目逐渐减少,7d兴奋性损伤组神经节细胞的数目更少,而且内丛状层变薄,与正常组比较差异有统计学意义(P0.05)。(2)PBS空白对照组：与正常对照组比较四个时间点均未见明显异常,差异无统计学意义(P0.05)。 2、视网膜电生理(ERG)检查：造模后3、7天,(1)NMDA实验组：与正常对照组比较,在标准混合反应中b波波幅显著下降,差异显著有统计学意义(P0.05)。(2)PBS空白对照组：与正常组比较没有明显差异(P0.05)。 3、TUNEL凋亡细胞染色：(1)NMDA实验组：四组神经节细胞层都可以见到红染的凋亡细胞,24h组凋亡细胞数量最多,与正常组比较差异有统计学意义(P0.05)。(2)PBS空白对照组：偶见少量凋亡细胞,与正常组比无统计学意义(P0.05)。 4、免疫组织化学：三组中都能见到GLAST蛋白的表达,正常组与PBS组可见GALST蛋白分布在视网膜全层,且主要分布在内外丛状层。NMDA组：四个时间点GLAST蛋白表达都明显减少。3、7d组能见到GALST蛋白在内丛状层、神经节细胞层的浓度要明显高于其他各层。 5、Taqman法荧光定量RT-PCR:(1)NMDA组：与正常组比较GLAST的mRNA表达显著下调,差异具有统计学意义(P0.05)。(2)PBS组：与正常组比较未见明显差别。结论： 1、玻璃体腔注射NMDA制作动物视网膜兴奋性损伤模型简便易操作、可重复性强,无论从形态学还是功能上都证明其可以用做理想的青光眼动物模型。 2、GLAST蛋白、mRNA表达下调,而神经节细胞存活率也下降,这说明GALST表达减少可能加重了神经节细胞的损伤。 3、GLAST蛋白表达发生了位置的重新分布,可能是一种自我保护的代偿机制。
[Abstract]:Aim: to observe the expression of glutamate transporter GLAST protein and the expression of mRNA over time in the excitatory injury of mouse retina induced by NMDA-N-methyl-D-aspartate (NMDA-D-aspartate), and to investigate the effect of GLAST on the apoptosis of ganglion cells. Methods: a total of 54 healthy C57BL / 6J mice of 4-8 weeks old, male and female, weighing about 20-30 g, were randomly divided into three groups: group A (n = 6) and control group (n = 24). Group B, NMDA excitatory injury group, group C, blank control group. Group B, group C, were killed at four time points after 6 hours, 24 hours and 7 days, respectively. Dark adaptation was carried out in each group at least 6 hours before death. The animals were killed after the operation of ERG. Each mouse's left eye was taken for pathological section, HE staining, immunohistochemical staining and TUNEL apoptotic cell staining were performed. The expression of glutamate transporter (GLASTmRNA) in the retina was detected by Taqman fluorescence quantitative RT-PCR method and compared with the normal group. Results 1in the NMDA excitatory injury group, there was no significant difference between the two groups at 6 hours after the model was made, compared with the normal control group. 24 hours after modeling, apoptosis began to appear in the ganglion cell layer of the retina. After 3 days, the number of apoptotic cells increased and the number of ganglion cells gradually decreased. In the group of excitatory injury for 7 days, the number of ganglion cells decreased, and the inner plexiform layer became thinner. Compared with the normal group, the difference was statistically significant (P 0.05). The PBS blank control group: compared with the normal control group, there was no obvious abnormality at the four time points, and the difference was not statistically significant (P 0.05). 2, ERG: the experimental group of NMDA: compared with the normal control group, the amplitude of b wave decreased significantly in the standard mixed reaction, and the difference was statistically significant (P 0.05). There was no significant difference between the control group and the normal group (P 0.05). 3Tunel apoptotic cells staining: in the experimental group, the number of apoptotic cells in the red stained apoptotic cells group was the highest in all the ganglion cell layers of the four groups, compared with the normal group, there was a significant difference between the two groups in the number of apoptotic cells in the control group: a small number of apoptotic cells were occasionally found in the control group, and a small number of apoptotic cells were occasionally found in the control group. There was no significant difference between the control group and the control group (P 0.05). 4Immunohistochemistry: the expression of GLAST protein could be seen in all three groups, and GALST protein could be seen in the whole retinal layer in normal group and PBS group. The expression of GLAST protein in the inner plexiform layer and the ganglion cell layer was significantly higher than that in the other layers at the four time points, and the expression of GALST protein in the inner plexiform layer was significantly decreased. 5Taqman fluorescence quantitative RT-PCR:(1)NMDA group: compared with the normal group, the mRNA expression of GLAST was significantly down-regulated, and the difference was statistically significant (P 0.05). There was no significant difference between the two groups. Conclusion:. 1. The animal model of retinal excitatory injury made by intravitreal injection of NMDA was simple, easy to operate and reproducible. It was proved to be an ideal animal model of glaucoma both in morphology and function. 2the expression of GLAST mRNA was down-regulated and the survival rate of ganglion cells was decreased, which suggested that the decrease of GALST expression might aggravate the injury of ganglion cells. 3 the expression of GLAST protein was redistributed, which may be a compensatory mechanism of self-protection.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R775

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