抑郁大鼠海马神经元结构改变及机制研究

发布时间：2018-04-28 09:02

本文选题：抑郁症 + 海马　；参考：《华北理工大学》2015年硕士论文

【摘要】：目的利用慢性不可预见性温和应激(chronic unpredicted mild stress,CUMS)建立大鼠抑郁症模型,通过观察微管相关蛋白p MAP-2和p-Tau在抑郁症大鼠海马的蛋白表达变化情况,进而探讨其与抑郁症的关系和意义。方法健康雄性SPF级SD大鼠80只,(体质量180~220g)随机的分成对照组与模型组(n=40),模型组给予慢性不可预见性温和应激(CUMS)建立模型。应激包括以下11种:潮湿垫料中24 h、进行行为束缚24 h、鼠笼进行45°倾斜24h、4℃的冰水游泳进行5 min、禁水24 h、禁食24 h、42℃热水游泳进行5 mi n、给予双耳电击5 s两次(0.8 m A)、夹尾1 min、进行鼠笼晃动15 min、持续光照明24 h。以上11种应激随机的进行分配,每天给予大鼠1种应激刺激,同种应激刺激不连续出现,共计两轮。对照组大鼠除每天抓取一次外,不做特殊处理。在应激刺激结束后分别于1、7、14和28d处死大鼠,对照组大鼠处死时间和模型组相同。分别通过糖水偏好实验、旷场实验以及Morris水迷宫实验对对照组和模型组大鼠进行行为学的检测;尼氏染色观察大鼠的海马神经元形态学;透射电镜观察大鼠海马神经元细胞超微结构,特别是微管结构的变化;采用免疫荧光双标法观察大鼠海马组织中p MAP-2和p-Tau共定位表达;采用Western blotting方法检测大鼠海马组织中p MAP-2和p-Tau蛋白表达水平。采用SPSS17.0统计软件对数据进行统计分析,以P0.05表示差异有统计学意义。结果糖水偏好实验:对照组和模型组大鼠普通水消耗量无明显变化,模型组大鼠糖水消耗量和糖水偏好百分比分别低于对照组,差异具有统计学的意义。旷场实验:模型组大鼠行走的总路程、中央格活动的时间、直立的次数和修饰行为次数少于对照组(P0.01)。Morris水迷宫实验:模型组大鼠的平均逃避潜伏期高于对照组(P0.01)。尼氏染色:结果表现为对照组的大鼠海马细胞密度大,锥体细胞层厚,排列紧密整齐,胞体饱满,突起显明。模型组大鼠的海马细胞密度变小,锥体细胞层变薄,细胞间隙变大,排列分布疏松,细胞核萎缩,突起相对减少。透射电镜:结果表现为对照组大鼠海马的神经细胞核膜较为完整,染色质结构相对正常,超微结构正常。模型组海马的细胞核相对固缩,核异染色质增多,发现一些小而不规则凝块、边缘聚合,内质网肿胀,线粒体嵴消失、肿胀、空泡化,还有细胞器减少、结构遭到破坏,以及在胞浆中发现形成大小不等的空泡。免疫荧光双标结果:各组大鼠海马区p MAP-2、p-Tau检测结果:p MAP-2为Alexa Fluor 488激发的绿色荧光,p-Tau为Alexa Fluor 594激发的红色荧光,图像重叠后可见部分神经元的胞浆内p MAP-2和p-Tau同时表达,表明p MAP-2和p-Tau有部分共定位。Western blotting检测结果:根据条带分析显示与对照组比较,p MAP-2蛋白和p-T au蛋白分别在CUMS后1d蛋白表达低于对照组(P0.01),7 d后开始升高(P0.01),14 d明显高于对照组(P0.01),28 d仍略高于对照组(P0.05)。结论1成功建立抑郁症的动物模型。2抑郁模型大鼠海马神经元发生变化。3微管蛋白p MAP-2和p-Tau蛋白表达水平在抑郁模型大鼠海马先降低后增高变化,这种变化可能影响微管的稳定性,破坏细胞骨架稳态,从而影响海马神经元的结构改变。
[Abstract]:Objective to establish a rat model of depression with chronic unpredictability and mild stress (chronic unpredicted mild stress, CUMS), and to explore the relationship and significance of the protein expression of microtubule related protein P MAP-2 and p-Tau in the hippocampus of depressive rats, and to explore the relationship and significance of the protein expression of P MAP-2 and p-Tau in the hippocampus of the depressive rats. Methods 80 healthy male SPF SD rats, (physique) 180~220g) was randomly divided into the control group and the model group (n=40). The model group was given a model of chronic unpredictability and mild stress (CUMS). The stress included 11 kinds: 24 h in the damp mat, 24 h for behavior, 45 degrees in the squirrel cage, 5 min for swimming at 4 C, 24 h, 24 h, and 5 Mi n in hot water at 42. The double ear electric shock was given 5 s two times (0.8 m A) and tail 1 min, the squirrel cage sloshing 15 min, and 11 kinds of stress above 24 h. in the continuous light illumination were randomly assigned. The rats were given 1 stress stimuli every day. The same stress irritation was discontinuous in two rounds. The control group was taken once a day and did not do special treatment. After the end of the stress stimulation, the rats were not treated with special treatment. The rats were killed in 1,7,14 and 28d respectively. The rats in the control group were killed at the same time as the model group. The behavior of the rats in the control group and the model group were detected by the sugar water preference experiment, the open field experiment and the Morris water maze experiment. The hippocampus neuron morphology was observed by Nissl staining, and the hippocampal neurons were observed by transmission electron microscope. The ultrastructure of the cell, especially the microtubule structure, was used to observe the co localization of P MAP-2 and p-Tau in the hippocampus of rats by immunofluorescence, and the Western blotting method was used to detect the expression of P MAP-2 and p-Tau protein in the hippocampus of rats. The data were statistically analyzed by the SPSS17.0 statistical software, and the difference was expressed by P0.05. Results the sugar water preference experiment: the normal water consumption of the control group and the model group had no obvious change. The sugar water consumption and the sugar water preference percentage in the model group were lower than those of the control group. The difference was statistically significant. The open field experiment: the total distance of walking in the model group, the time of the central grid activity, the erect times and the number of the rats in the model group. The number of modified behavior was less than that of the control group (P0.01).Morris water maze experiment: the average escape latency of the model group was higher than that of the control group (P0.01). Nissl staining: the results showed that the hippocampus cell density of the control group was large, the pyramidal cell layer was thick, the cell body was full and the protuberance was obvious. The density of the hippocampus cells in the model group became smaller. The pyramidal cell layer became thinner, the cell space became larger, the distribution was loose, the nucleus atrophied and the protuberance was relatively reduced. Transmission electron microscope showed that the nuclear membrane of the hippocampus of the control group was more complete, the chromatin structure was relatively normal, and the ultrastructure was normal. The nucleus of the model group was relatively condensed, and the nuclear heterochromatin was increased, found that the nuclear heterochromatin was on the increase, found that the nucleus of the nucleus increased. Small and irregular clot, edge polymerization, endoplasmic reticulum swelling, mitochondrial crista disappearance, swelling, vacuolization, organelle reduction, structure destruction, and the discovery of vacuoles in the cytoplasm. Double immunofluorescence results: P MAP-2, p-Tau test results in hippocampus of rats in each group: P MAP-2 for Alexa Fluor 488 green fluores. Light, p-Tau is the red fluorescence excited by Alexa Fluor 594, and the P MAP-2 and p-Tau in the cytoplasm of the visible part of the neurons are expressed at the same time after the image overlap. It shows that P MAP-2 and p-Tau have a partial CO localization of.Western blotting detection results. In the control group (P0.01), after 7 d (P0.01), 14 d was significantly higher than that of the control group (P0.01), and 28 d was still slightly higher than the control group (P0.05). Conclusion 1 successfully established the animal model of depression, the hippocampus neurons of the depressive model of.2, the.3 microtubule P MAP-2 and the expression of the p-Tau protein expression in the hippocampus of the depressive model rats increased and then increased. This change may affect the stability of microtubules and destroy the homeostasis of cytoskeleton, thus affecting the structural changes of hippocampal neurons.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R749.4

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