GSK-3β、CRMP-2在形觉剥夺性弱视大鼠视皮层的表达
发布时间：2018-01-26 02:55
本文关键词： 糖原合成酶激酶3β 脑衰反应调节蛋白2 形觉剥夺性弱视 大鼠 视皮层 出处：《南华大学》2016年硕士论文 论文类型：学位论文
【摘要】：目的:研究糖原合成酶激酶3β(Glycogen synthase kinase 3β,GSK-3β)和脑衰反应调节蛋白2(Collapsin response mediator protein 2,CRMP-2)在形觉剥夺性弱视大鼠视皮层中的表达,探索其在弱视发病机制中的意义。方法:1.出生14天的健康大鼠64只,随机选择32只大鼠行单侧眼睑缝合术作形觉剥夺性弱视组,另外32只大鼠不做任何处理成为正常组。根据大鼠出生的天数,在21天时将形觉剥夺性弱视组和正常组大鼠用10%水合氯醛麻醉后行P-VEP(Pattern Visual Evoked Potential)检测,记录P100波的潜伏期和波幅值,判断单眼形觉剥夺性弱视模型是否建立成功。2.根据大鼠出生的天数,分别在14天、21天、45天、120天4个时间点处死形觉剥夺性弱视组和正常组大鼠,每小个时间点处死大鼠各8只。所有处死的大鼠均取其大脑视皮层组织,用4%多聚甲醛PBS溶液固定后,封蜡行组织切片。采用HE(Hematoxylin Eosin)染色观察形觉剥夺性弱视组和正常组大鼠视皮层神经元细胞的形态;然后采用sp免疫组化法检测形觉剥夺性弱视组和正常组大鼠视皮层4个时间点gsk-3β、crmp-2免疫阳性神经元的表达,并使用图像分析系统测定gsk-3β、crmp-2免疫阳性神经元表达的平均光密度值(aod值)并进行统计学分析。结果:1.p-vep检测:与正常组相比,形觉剥夺性弱视组大鼠p100波的潜伏期明显延长(p0.05),振幅明显降低(p0.05),提示单侧眼睑缝合7天后,可成功建立单眼形觉剥夺性弱视大鼠模型。2.he染色:正常组大鼠视皮层神经元细胞轮廓清晰,可清楚显示各神经元细胞的形态,细胞分布较密集,形觉剥夺性弱视组大鼠视皮层神经元分布稀松,且排列紊乱,无法清晰辨别细胞形态,胞体变小,核固缩,呈嗜碱性,部分核溶解。3.免疫组化结果:1)gsk-3β在大鼠视皮层存在表达。正常组大鼠视皮层gsk-3β的表达在14天,21天,45天,120天各时间点之间比较,各时间点之间均无明显差异(p0.05)。形觉剥夺性弱视组大鼠视皮层gsk-3β的表达在14天,21天,45天,120天各时间点之间比较,除45天与120天比较无差异(p0.05)外,其余各时间点之间两两比较均有差异(p0.05)。与正常组大鼠相比,形觉剥夺性弱视大鼠视皮层gsk-3β的表达在14天时无明显差异(p0.05),但在21天,45天,120天时均升高(p0.05)。2)crmp-2在大鼠视皮层中存在表达。正常组大鼠视皮层crmp-2的表达在14天,21天,45天,120天各时间点之间比较,除45天与120天比较无差异(p0.05)外,其余各时间点之间两两比较均有差异(p0.05)。形觉剥夺性弱视组大鼠视皮层crmp-2的表达在14天,21天,45天,120天各时间点之间比较,除45天和120天(p0.05)外,其余各时间点之间两两比较均有差异均有统计学意义(p0.05)。与正常组大鼠相比,形觉剥夺性弱视大鼠视皮层crmp-2的表达在14天时无明显差异(p0.05),但在21天,45天,120天时均降低(p0.05)。3)大鼠视皮层gsk-3β和crmp-2表达的相关性:在正常组大鼠视皮层中gsk-3β和crmp-2两者之间无明显相关性(r=-0.142,p0.05),而在形觉剥夺性弱视组大鼠视皮层中,两者呈负相关性(r=-0.838,p0.05)。结论:1.在大鼠视觉发育关键期的早期,行单侧眼睑缝合术7天后可以成功建立单眼形觉剥夺性弱视大鼠模型。2.在正常大鼠视觉发育关键期内,gsk-3β和crmp-2在大鼠大脑视皮层均有表达,说明二者有可能参与大鼠视觉发育的过程。3.在形觉剥夺性弱视大鼠视皮层中,gsk-3β的表达随着剥夺时间的延长逐渐升高,45天时达到峰值,而crmp-2的表达随着剥夺时间延长而逐渐下降,45天时达到低值,两者呈负相关性,提示在视觉发育的可塑性期内,gsk-3β和crmp-2可能通过某种调节机制参与弱视的发生发展过程。
[Abstract]:Objective: To study the influence of glycogen synthase kinase 3 (Glycogen synthase kinase 3 beta, beta GSK-3) and collapsin response mediator protein 2 (Collapsin response mediator protein 2, CRMP-2) in the perception of visual expression in the cortex of deprived amblyopia rats, and explore its significance in the pathogenesis of amblyopia. Methods: 1. 14 days of birth 64 healthy rats, 32 rats were randomly selected for unilateral eyelid suture for deprivation amblyopia, another 32 rats without any treatment as normal group. According to the number of days the rats were born, and in 21 days will form deprivation amblyopia group and normal group rats 10% chloral hydrate anesthesia (P-VEP Pattern Visual Evoked Potential) detection, record the latency and amplitude of P100 wave value judgment of monocular deprivation amblyopia model is established successfully according to the number of days.2. rats were born, respectively in 14 days, 21 days, 45 days, 120 days and 4 at the time of death Form deprivation amblyopia group and normal group rats, each time the rats were sacrificed 8 rats each. All rats were sacrificed were removed from the visual cortex tissue, with 4% paraformaldehyde fixed PBS solution after wax for tissue sections. By HE (Hematoxylin Eosin) staining of form deprivation amblyopia group and normal group rat visual cortex neuronal morphology; then using SP immunohistochemical method to detect the form deprivation amblyopia group and normal group rat visual cortex at 4 time points of GSK-3 beta, the expression of crmp-2 immunoreactive neurons, and use image analysis system for determination of GSK-3 beta, the average optical density of crmp-2 immunoreactivity positive neurons value (AOD value) and analyzed statistically. Results: 1.p-vep detection: compared with the normal group, deprivation amblyopia rats significantly prolonged the latency of P100 wave (P0.05), the amplitude decreased (P0.05), suggesting that unilateral eyelid suture can be 7 days. The successful establishment of monocular deprivation amblyopia rats.2.he staining: normal rat visual cortical neuron outline clear, can clearly show the neuron cell morphology, cell distribution is dense, the perception of visual cortical neurons were distributed deprived amblyopia rats, and unable to clearly identify the disorder, cell morphology, cell the body becomes small, karyopyknosis, basophilic nucleus dissolved.3. immunohistochemical results: 1) GSK-3 beta in the visual cortex of rats. The expression of GSK-3 beta expression in the cerebral cortex as normal rats in 14 days, 21 days, 45 days, 120 days between each time point, each time some were not significantly different (P0.05). The expression of GSK-3 beta form sense visual cortex deprived amblyopia rats in 14 days, 21 days, 45 days, 120 days between each time point, in addition to 45 days compared with 120 days of no difference (P0.05), 22 were the differences between the other time points (P0.05) and positive. 甯哥粍澶ч紶鐩告瘮,褰㈣鍓ュず鎬у急瑙嗗ぇ榧犺鐨眰gsk-3尾鐨勮〃杈惧湪14澶╂椂鏃犳槑鏄惧樊寮，
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