EphrinB3蛋白在皮层发育障碍致痫大鼠海马区表达的研究

发布时间：2018-05-29 08:25

本文选题：癫痫 + 皮层发育障碍　；参考：《中南大学》2014年硕士论文

【摘要】：目的本研究通过建立皮层发育不良模型与匹罗卡品癫痫大鼠模型,检测皮层发育障碍致痫的大鼠及匹罗卡品癫痫大鼠模型中海马区EphrinB3蛋白的表达变化,探讨其在皮层发育不良致痫过程中的作用。方法1.卡莫司汀诱导皮层发育障碍大鼠模型的建立及评估：10只SD孕鼠在怀孕17(E17)天腹腔内注入卡莫司汀(Carmustine, BCNU)(15mg/Kg),所产仔鼠入模型组；9只SD孕鼠在E17天腹腔内注入5%葡萄糖水(15mg/Kg),所产仔鼠入对照组。观察皮层发育畸形仔鼠生长发育、脑湿重及仔鼠脑组织苏木素-伊红(HE)染色。 2.匹罗卡品癫痫模型的建立：随机从皮层发育不良组及对照组抽取P60天雄鼠建立匹罗卡品癫痫模型,制备皮层发育障碍癫痫组(MCD+EP组)、普通大鼠癫痫组(EP组)、皮层发育障碍组(MCD组)及对照组(CON组)。 3.分别比较模型组和对照组大鼠的癫痫发生的潜伏期、癫痫持续状态(SE)持续的时间及癫痫发生的死亡率,评估两组大鼠的癫痫易感性。 4.采用免疫组化、免疫荧光及Real-time PCR分析方法检测各组在SE诱导成功后1天(急性期)、14天(静止期)及60天(慢性期)三个时间点大鼠海马齿状回中EphrinB3蛋白及mRNA的表达变化。结果1.皮层发育障碍模型仔鼠一般状态较对照组差。脑组织HE染色示模型组仔鼠的大脑皮质变薄、皮质及海马层状结构紊乱,皮质发育不良模型成功。 2.在MCD基础上通过氯化锂-匹罗卡品建立匹罗卡品癫痫大鼠动物模型,建立皮层发育不良癫痫大鼠动物模型。MCD模型鼠注射匹罗卡品后,SE发生的潜伏期显著缩短(P0.05),癫痫持续状态时间延长(P0.01),成功率及死亡率增加(P0.01),提示皮层发育障碍大鼠的癫痫易感性增加。 3.免疫组化与免疫荧光结果表明EphrinB3蛋白主要分布于海马齿状回；与EP组比较,MCD+EP组的EphrinB3在海马齿状回的表达显著上调,差异具有显著性(P0.01),并在急性期、静止期表达下调,慢性期逐渐表达上调。与CON组比较,EP组在慢性期的EphrinB3表达也显著上调(P0.01)。 4. Real-time PCR结果表明MCD+EP组EphrinB3mRNA的表达水平较EP组明显增高,并在急性期、静止期表达下调,慢性期表达逐渐上调。结论1. EphrinB3蛋白可能参与了皮层发育障碍大鼠海马区异常神经网络的兴奋性调节。 2. EphrinB3蛋白可能使皮层发育障碍大鼠对癫痫的易感性增高。
[Abstract]:Objective to study the changes of EphrinB3 protein expression in hippocampus of epileptic rats induced by cortical dysplasia and pilocarpine epileptic rats by establishing cortical dysplasia model and pilocarpine epileptic rat model. To explore its role in the process of eclampsia caused by cortical dysplasia. Method 1. Establishment and Evaluation of Cumoxetin-induced Cortical dysplasia in Rats: 10 Sprague-Dawley pregnant mice were intraperitoneally injected with Carmustine Carmosine and 15 mg / kg KgCU on gestational day 17. 9 SD pregnant rats in the model group were injected with 5% glucose water 15 mg / kg / L intraperitoneally on 17 days after gestation. The pups were added into the control group. Growth and development, brain wet weight and hematoxylin-eosin (HEH) staining were observed. 2. Establishment of pilocarpine epilepsy model: P60 day male rats were randomly selected from cortical dysplasia group and control group to establish pilocarpine epilepsy model. Preparation of cortical dysplasia epilepsy group MCD EP group, normal rat epilepsy group, cortex dysplasia group (MCD group) and control group (Con group). 3. The latency, duration and mortality of epileptogenesis were compared between the model group and the control group, and the susceptibility to epilepsy of the two groups was evaluated. 4. Immunohistochemistry, immunofluorescence and Real-time PCR analysis were used to detect the expression of EphrinB3 protein and mRNA in dentate gyrus of rats at 1 day after SE induction (14 days in acute phase (stationary phase) and 60 days in chronic phase). Result 1. The general state of the model mice with cortical dysplasia was worse than that of the control group. Brain tissue HE staining showed that cerebral cortex thinned, cortex and hippocampus lamellar structure disorder, cortical dysplasia model was successful in the model group. 2. On the basis of MCD, a rat model of pilocarpine epilepsy was established by lithium-pilocarpine chloride. Establishment of Cortical dysplasia of Epilepsy Rat Model .MCD Model Rats after injection of pilocarpine, the latency of SE was significantly shortened, the duration of epileptic status was prolonged, and the success rate and mortality were increased (P 0.01), indicating that the rats with cortical dysplasia had a significant increase in the latency of SE, which indicated that the rats with cortical dysplasia had a significant increase in the rate of success and mortality. Increased susceptibility to epilepsy. 3. Immunohistochemical and immunofluorescence results showed that EphrinB3 protein was mainly distributed in the dentate gyrus of Yu Hai horses, and the expression of EphrinB3 in the dentate gyrus was significantly up-regulated in the MCDEP group compared with the EP group, the difference was significant (P 0.01), and the expression of MCDEP was down-regulated in the acute phase and the stationary phase. The expression of chronic phase was gradually up-regulated. Compared with CON group, the expression of EphrinB3 was also significantly up-regulated in EP-treated group in chronic phase. 4. The results of Real-time PCR showed that the expression of EphrinB3mRNA in MCD EP group was significantly higher than that in EP group, and the expression of EphrinB3mRNA was down-regulated in acute phase, resting stage, and gradually up-regulated in chronic phase. Conclusion 1. EphrinB3 protein may be involved in excitatory regulation of abnormal neural network in hippocampus of rats with cortical dysplasia. 2. EphrinB3 protein may increase the susceptibility to epilepsy in rats with cortical dysplasia.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

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