本文關鍵詞： 谷氨酸 谷氨酸轉運體 GLAST 視神經保護 神經節(jié)細胞　出處：《青島大學》2013年碩士論文　論文類型：學位論文

【摘要】：目的：本實驗在NMDA(N-甲基-D天門冬氨酸)所致小鼠視網膜興奮性損傷中,觀察谷氨酸轉運體GLAST的蛋白表達量和mRNA隨時間表達量的變化,探討GLAST對神經節(jié)細胞凋亡的作用。 方法：本實驗采用健康清潔級4-8周齡C57BL/6J小鼠共54只,雌雄各半,體重約20-30g,隨機分成A、B、C三組,A組6只為正常對照組,B、C為實驗組各24只,其中B組為NMDA興奮性損傷組,C組為空白對照組。B組、C組于造模后6h、24h、3d和7d四個時間點處死。處死前對每組小鼠進行暗適應,至少暗適應6小時,然后進行視網膜電生理(ERG)檢查。ERG操作完畢處死動物,取每只小鼠左眼眼球做病理切片,進行HE染色、免疫組織化學及TUNEL凋亡細胞染色,右眼眼球取完整視網膜用Taqman熒光定量RT-PCR法檢測視網膜中谷氨酸轉運體GLASTmRNA隨時間表達變化,以及與正常組比較表達差異。 結果1、HE染色：(1) NMDA興奮性損傷組：造模后6h,與正常對照組比較未見明顯差異,造模后24h,視網膜的神經節(jié)細胞層開始出現(xiàn)凋亡形態(tài)細胞,3d凋亡細胞增多并且神經節(jié)細胞數(shù)目逐漸減少,7d興奮性損傷組神經節(jié)細胞的數(shù)目更少,而且內叢狀層變薄,與正常組比較差異有統(tǒng)計學意義(P0.05)。(2)PBS空白對照組：與正常對照組比較四個時間點均未見明顯異常,差異無統(tǒng)計學意義(P0.05)。 2、視網膜電生理(ERG)檢查：造模后3、7天,(1)NMDA實驗組：與正常對照組比較,在標準混合反應中b波波幅顯著下降,差異顯著有統(tǒng)計學意義(P0.05)。(2)PBS空白對照組：與正常組比較沒有明顯差異(P0.05)。 3、TUNEL凋亡細胞染色：(1)NMDA實驗組：四組神經節(jié)細胞層都可以見到紅染的凋亡細胞,24h組凋亡細胞數(shù)量最多,與正常組比較差異有統(tǒng)計學意義(P0.05)。(2)PBS空白對照組：偶見少量凋亡細胞,與正常組比無統(tǒng)計學意義(P0.05)。 4、免疫組織化學：三組中都能見到GLAST蛋白的表達,正常組與PBS組可見GALST蛋白分布在視網膜全層,且主要分布在內外叢狀層。NMDA組：四個時間點GLAST蛋白表達都明顯減少。3、7d組能見到GALST蛋白在內叢狀層、神經節(jié)細胞層的濃度要明顯高于其他各層。 5、Taqman法熒光定量RT-PCR:(1)NMDA組：與正常組比較GLAST的mRNA表達顯著下調,差異具有統(tǒng)計學意義(P0.05)。(2)PBS組：與正常組比較未見明顯差別。 結論： 1、玻璃體腔注射NMDA制作動物視網膜興奮性損傷模型簡便易操作、可重復性強,無論從形態(tài)學還是功能上都證明其可以用做理想的青光眼動物模型。 2、GLAST蛋白、mRNA表達下調,而神經節(jié)細胞存活率也下降,這說明GALST表達減少可能加重了神經節(jié)細胞的損傷。 3、GLAST蛋白表達發(fā)生了位置的重新分布,可能是一種自我保護的代償機制。
[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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R775

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