【作者】 冯占辉；

【导师】 晏勇；

【作者基本信息】 重庆医科大学 ， 神经病学， 2010， 博士

【摘要】 第一部分X射线照射建立大鼠皮质发育障碍模型研究目的:应用X射线建立大鼠皮质发育障碍模型,筛选最佳放射剂量模型。方法:应用X射线给妊娠17天的Sprague-Dawley (SD)大鼠不同放射剂量照射。根据放射剂量分为5组:模型①组125cGy、模型②组150cGy、模型③组175cGy、模型④组200cGy,模型⑤组225cGy,同时设立对照组。观察指标:①统计各组仔鼠的存活率,观察仔鼠一般行为学特点。②检测不同时间点仔鼠体重变化。③P28d采用Morris水迷宫实验测试各组仔鼠学习和空间记忆能力。④P56d处死仔鼠观察大脑形态、测量脑湿重,常规病理检查。⑤P63d用红藻氨酸诱导各组仔鼠癫痫发作,比较其痫性发作阈值和死亡率。选择存活率高、精神发育迟滞明显、痫性发作阈值降低、有典型神经元异位结节病理改变的大鼠组为最佳剂量模型。结果:1)对照组与模型①、②、③、④和⑤各组仔鼠的存活率分别为96.8%、100%、93.5%、93.5%、50%、20%。行为学观察:对照组、①组和②组未发现行为异常。③组易激惹,活动量少,反应迟钝,摄食和饮水正常。④组仔鼠活动量减少、轻度嗜睡,并有明显反应迟钝、灵活性降低等表现,摄食和饮水减少,未见痫性发作。2)模型①、②组仔鼠出生后不同时间点体重与对照组比较无差异(P>0.05)。模型③组仔鼠P0d和P14d体重较对照组仔鼠体重下降(P<0.05),在P28d和P56d与对照组同时间点比较无差异(P>0.05)。模型④组仔鼠出生后不同时间点体重均低于对照组体重(P<0.05)。模型⑤组仔鼠体重各时间点较对照组比较明显降低(P<0.05),并随着鼠龄增长全部死亡。3) P28d水迷宫实验显示①组仔鼠和对照组相比逃避潜伏期及跨过原平台次数无明显差异(P>0.05)。模型②组仔鼠逃避潜伏期在最初的第1天和第2天延长,第3天和第4天水中逃避潜伏期与对照组无统计学差异(P>0.05)。模型③和④组仔鼠在逃避潜伏期及跨过原平台次数较对照组明显延长(P<0.05)。4) P56d处死对照组和模型组仔鼠各6只,模型②③组大脑重量与对照组比较降低(P<0.05)。模型④组仔鼠脑组织重量显著减轻、体积显著减小(P<0.05)。模型③④组仔鼠大脑皮质弥漫性皮质发育不良伴神经元异位结节发生率为100%。典型病理改变包括大脑皮质厚度变薄、层状结构紊乱、神经元缺失,皮质及海马可见神经元异位结节等,类似人类皮质发育不良伴神经元异位结节。5) P63d红藻氨酸诱发癫痫发作实验中,模型②③组仔鼠痫性发作的潜伏期缩短,但癫痫持续状态时间与对照组比较无显著差异(P>0.05)。④组仔鼠痫性发作的潜伏期缩短、阈值降低,癫痫持续状态较对照组时间延长(P<0.05),死亡率明显高于对照组(P<0.05),主要以Ⅴ级强直阵挛发作为主。结论:1. X射线是制作DCDs模型鼠的良好手段。2. 175cGy剂量组可以制作出理想的仔鼠DCDs模型,该模型生存率高,能够很好模拟人类智能发育迟滞,容易诱发痫性发作,具有典型的DCDs伴神经元异位结节病理改变。第二部分皮质发育障碍模型鼠与水脑畸形儿童大脑影像学对比研究目的:尝试影像学评价放射线DCDs模型;探讨模型鼠和水脑畸形儿童头颅影像学特点,并进行对比分析。方法:应用直线加速器175cGy剂量照射孕17天SD大鼠,待仔鼠满P56时行鼠脑磁共振扫描。扫描后处死,取脑进行组织病理学研究。临床方面收集儿童水脑畸形病例,应用磁共振进行头颅平扫。将模型鼠脑影像与水脑畸形儿童临床和影像学结果进行对比分析。结果:1)DCDs模型大鼠头颅MRI特点:脑体积略小,皮层厚度变薄,双侧侧脑室、第三、第四脑室明显扩大,胼胝体变小伴有缺如,四叠体明显暴露,小脑体积变大。2)水脑畸形头颅MRI特点:颅脑扩大,第三、第四及双侧侧脑室扩大,全脑叶皮质变薄或缺如,胼胝体变小甚至缺如。3)对比结果:共同点为均有第三脑室、第四脑室、双侧侧脑室的扩大,皮层变薄,胼胝体变小甚至缺如。区别点为积水型无脑畸形的皮层损害严重,脑积水更加严重。结论: 1.1.5T磁共振扫描能够很好反映DCDs模型鼠皮质发育不良的特点;2.该模型尚不能模拟人类水脑畸形。第三部分皮质发育障碍模型乳鼠海马基因芯片研究目的:应用基因芯片技术,探讨DCDs模型乳鼠和对照组乳鼠海马差异基因表达,以期发现模型乳鼠海马差异基因中与难治性癫痫密切相关的基因。方法:建立175cGyX射线诱导的DCDs大鼠模型,在P0d提取模型鼠和对照组海马组织总RNA,反转录为cDNA,采用体外转录方法获得cRNA与Affymetrix的大鼠表达谱芯片进行杂交。采用扫描仪进行扫描,处理数据。结果:X射线组DCDs模型乳鼠与对照组乳鼠海马差异基因如下:共发现149个差异表达基因,其中45个基因上调,104个基因下调。结合成年鼠蛋白质组学研究结果,其中与难治性癫痫密切相关的基因是生长相关蛋白43(GAP43),该蛋白可能参与苔藓纤维的芽生,异常网络的重构。突触小体相关蛋白25(SNAP25),该蛋白可能参与突触重塑,造成突触传导异常。两个基因表达均上调。结论:1.X射线模型乳鼠海马中GAP43和SNAP25基因表达上调;2.两个基因可能是参与海马苔藓纤维芽生和突触重塑的重要调节基因,两个基因的上调与DCDs易感癫痫密切相关。第四部分皮质发育障碍模型成年鼠海马蛋白质组学研究目的:采用双向凝胶电泳技术探讨DCDs模型成年鼠海马与对照组海马的差异蛋白表达,分析与难治性癫痫密切相关的靶蛋白。方法:应用X射线175cGy剂量制作DCDs模型鼠,从P56d成年模型鼠和对照组中随机选择6只进入实验。将大鼠断头取脑,提取海马并测蛋白浓度,然后进行双向电泳获得凝胶图谱,应用PDQUEST软件进行图像分析,寻找差异蛋白点,对差异蛋白点进质谱鉴定,查询后获得差异表达蛋白。结果:①成功获得分辨率高和重复性好的模型组和对照组大鼠海马组织蛋白凝胶图谱。②经质谱鉴定最终获得8个差异蛋白点,GTP结合蛋白,突触相关蛋白90,突触囊泡蛋白,钙网蛋白等4个上调;肌酸激酶,Cu-Zn超氧化物歧化酶,二氢密啶相关蛋白、谷胱甘肽S-转移酶等4个下调。结论:1.利用双向电泳技术可以获得较为理想的二维凝胶电泳图谱,双向电泳技术是适合做蛋白质组学分析的。2.X射线模型鼠海马与对照组海马存在明显差异蛋白表达,其中与突触重塑和苔藓纤维芽生相关蛋白可能与癫痫易感性密切相关。更多还原

【Abstract】 PART ONE: RESEARCH OF THE MODELS WITH DCDCS INDUCED BY X-RAYObjective:To establish animal models with disorders of cortical development (DCDs) by x-ray and select the optimal dose of the established modle radiated by x-ray.Methods : Pregnant Sprague-Dawley rats were given x-ray with different radiation doses at embryonic day 17. There are five doses groups :the group1 125cGy, the group2 150cGy, the group3 175cGy, the group4 200cGy, the group5 225cGy, the control group 0 cGy .The following aspects were observed in all groups.①We calculated the survival rate of rat offspring in each group and observed their general state of behaviors.②The weight reflecting developmental condition of rat offsprings were measured at different time points.③We adopted Morris-water-maze experiment to test the learning and memory capacity of rat offsprings in each group on P28d.④After measuring the wet weight of brains and observing their appearance, we examined the changes of brain pathology in cerebral cortex and hippocampus to confirm the types of DCDs on P56d..⑤We used kainic acid to induce seizure and compared their seizure threshold and mortality induced by seizure on P63d. The classical model induced by x-ray should be of hign survival rate, mental retardation, lower threshold of epileptic seizure and typically pathologic change of DCDs.Results:1) The survival rate of rat offsprings in the control group and the five model groups were 96.8%, 100%, 93.5%, 93.5%, 50%,20%. respectively. During the development of rats, No abnormality existed in the control, the model 1 and the model 2. The rat offsprings in model 3 were irritable, mild somnolence, fewer activities and dullness. The rat offsprings in model 4 were fewer activities, somnolence,obvious dullness and reduction of drinking water and feeding. No obvious seizure was observed in the control and five groups.2) Weight difference were not found among the rat offsprings of the control group and the model 1 and model 2 (P>0.05). The mean weight of rat offsprings in the model 3 reduced slightly at P0d and P14d(P<0.05), but the difference of weight disappeared at P28d and P56d(P>0.05). The weight of the rat offsprings in model 4 reduced heavily at different point(P<0.05).3) Morris-water-maze experiment showed that the rat offspring in model 1 did not show any disparity with the control group on the escape latency and times of crossing platform (P>0.05). The escape latency of rat offsprings in model 2 were longer than that of control group on first day and second day, but the difference disappeared on third day and fourth day(P>0.05). The escape latency and times of crossing platform of rat offsprings in the model 3 and the model 4 were always longer than those of the control group(P<0.05).4) The six rat were killed by P56d among the control and the model group. Brain weight of rat offspring in model 2 and model 3 obviously reduced(P<0.05). Reduced weight and abnormal construction of brain were displayed in the model 4(P<0.05). The incidence rate of DCDs of offsprings in the model 3 and model 4 reached 100%. Typical pathological features included a thin cortical plate, disorder of layer structure, and distinct clusters of neuronal elements that represented heterotopias emerged in cerebral cortex and hippocampus. These morphologic features of DCDs found in this model were similar to cortical dysphasia and neuronal heterotopias in humans.5)The latent period of Kainic acid inducing seizure was shortened in the model 2 and the model 3(P<0.05), but the time of epileptic state and the mortality induced by seizure of rat offspring in the second group were the same as that of control group(P>0.05) in kainic acid inducing seizure experiment on P63d. The rat offspring in the model 4 had a significantly lower seizure threshold and longer time of epileptic state than those of control group, the mortality induced by seizure of rat offspring was obviously higher than that of control group(P<0.05).Conclusion:1. X-ray by which the DCDs model were made was the good method. 2. The model induced by 175cGy was of hign survival rate, mental retardation, lower threshold of epileptic seizure and typically pathologic changes of DCDs.PART TWO: COMPARATIVE STUDY OF THE IMAGE OF BRAIN BETWEEN THR RATS WITH DCDS AND THE CHILDREN WITH HYDRANENCEPHALYObjective:Try to apply 1.5Tesla MRI on model rats with DCDs and to investigate brain image feature of the model rats and the children with hydranencephaly. To analyse imaging data and make comparative anlysis.Methods:Time-pregnant SD rats were irradiated on embryonic day E17 with 175 cGy or were left untreated. At P56d, the animals were scaned 1.5Tesla MRI, then were killed and their brains processed for histological studies. We collected the cases with hydranencephaly. All patients were scaned by MRI. At last we make comparative anlysis between the rats with DCDs and the patients. Results:1) Brain image feature of the model rat showed that small brain volume, enlargement of both lateral cerebral ventricle, the third ventricle and the four ventricle, thinning cortex, thinning corpus callosum and exposure of quadrigeminal bodies. 2) Brain image feature of the hydranencephaly showed that brain expansion, enlargement of both lateral cerebral ventricle , the third ventricle and the four ventricle, thinning cortex or absence of cortex, thinning corpus callosum or absence of callosum. 3) The results of comparison showed that the same aspects were enlargement of both lateral cerebral ventricle and the third ventricle, thinning cortex, thinning corpus callosum. The different aspects were hydranencephaly with more severe damage of cortex and hydrocephalus malformation.Conclusion:1. MRI provides a new approach for diagnosis of DCDs. 2.The characteristic of image of rats with DCDs don’t simulate feature of hydranencephaly.PART THREE:RESEARCH OF THE GENE CHIP OF HIPPOCAMPUS IN LITTERS WITH DCDSObjective: :To obtain the differences expression gene among the hippocampus tissues between litters in the control group and litters with DCDs induced by x-ray and find the gene related intractable epilepsy in hippocampus in model litters. Methods: The animal model induced by 175cGy x-ray was established. The total RNAs were isolated from hippocampus tissues of newborn rats in Pod. cDNA were synthesized. Then,cRNAs which obtained by IVT hybrided with the rat affymetrix chip.The genechip were scaned by affymetrix scanner . The data were obtained and analyzed by software.Results: The different expression gene were shown : totally 149 differential expression gene including 45 upregulated genes and 104 downregulated gene. Based on the results of proteomics research, We found the gene related intractable epilepsy : GAP43 which may be involved in the sprouting of mossy fiber and reconstruction of nerve network.and SNAP 25 which may be involved in remolding of synapse. The expression of two gene was upregulated.Conclusions: 1. Expression of both GAP43 and SNAP25 gene were upregulated. 2. Both of them may be the key gene which were involved in the sprouting of mossy fiber and remolding of synapse to potentiate epileptic activities. Objective: To obtain the differences proteins of hippocampus between the animal model with DCDs induced by x-ray and the control and to find out the target and effective proteins related with intractable epilepsy.Methods : The animal model induced by 175cGy X-ray was established. 6 rat s were randomly selected in the model group and the control. We rapidly dissected the whole brain of each animal, extracted total protein of hippocampus and measure the protein concentration. Then the gel maps were obtained by 2D-E. The differential protein spots were analyzed by PDQUEST software. They will be identified by mass spectrometry. After consulting the NCBI, the different proteins were obtained.Results:①Well resolution and repetitiveness 2-DE gel images of rat brain proteome were obtained by 2D-E.②Eight different spots were obtained by consulting the NCBI, four upregulation proteins: GTP-binding protein,synapse-associated protein 90,synaptotagmin 8, calreticulin; four downregulation proteins: Cu-Zn superoxide dismutase ,dihydropyrimidinase-related protein , creatine kinase , glutathione S-transferase P.Conclusion: 1.The ideal brain 2D-E images could be obtained by 2D-E. 2.The obvious expression difference of hippocampus protein between the rats with DCDs and the control were found. The protein related with the sprouting of mossy fiber and remolding of synapse may play an important role to potentiate epileptic activities.更多还原