【作者】 吴隽松；

【导师】 邬丽莎；

【作者基本信息】 泸州医学院 ， 生理学， 2011， 硕士

【摘要】 目的:癫痫是由大脑神经元反复超同步放电致反复性惊厥发作为特征的一种神经系统常见疾病。癫痫发作能造成神经元损伤已被广泛认可,而另一方面多种外部因素的改变,比如癫痫、帕金森病等可以影响发育中的和成年啮齿类动物的神经元的产生。近年成年哺乳动物海马神经发生的调控机制的研究显示,局部微环境控制了神经发生,此认识在理解神经干细胞如何增殖、分化,最终整合到成熟的神经环路中有重要作用。黄芪甲苷是从黄芪中提取的有效单体成分,具有抗氧化、抗应激、抗炎、免疫调节、健脑益智等多种药理作用,故此实验希望进一步了解黄芪甲苷对大鼠癫痫模型(海人酸KA模型)是否具有促进海马细胞增殖作用基础上,进一步明确黄芪甲苷对在体癫痫大鼠的神经保护作用。方法:本研究将SD大鼠随机分为假手术组、模型组、黄芪甲苷干预组,应用立体定向脑室内微量注射的方法建立KA诱导的大鼠癫痫模型。采用Morris水迷宫实验观察大鼠学习记忆能力的影响;尼氏染色观察大鼠海马神经元的形态及数目改变;BrdU和免疫组织化学方法来观察大鼠海马齿状回颗粒细胞下层BrdU阳性细胞数目及海马齿状回区星形胶质细胞数目的变化;观察黄芪甲苷干预对海马细胞增殖的影响及对脑损伤后的保护作用,为进一步研究癫痫有效治疗方式提供动物实验依据。结果:1、Morris水迷宫实验(1)定位航行试验:各时间点第l天,假手术组与模型组、黄芪甲苷干预组比较有显著差异(p<0.01),而模型组与黄芪甲苷干预组比较无统计学意义(p>0.05);第2天,与假手术组的逃避潜伏期比较,模型组逃避潜伏期明显延长(p<0.05);黄芪甲苷干预组的逃避潜伏期较模型组无差异(P>0.05)。第3-5天,模型组大鼠的逃避潜伏期与假手术组及黄芪甲苷干预组比较逃避潜伏期明显延长(P<0.05);(2)空间探索试验结果:模型组大鼠120s内穿越平台区域的次数明显低于假手术组(P<0.01);黄芪甲苷干预组大鼠120s内穿越平台区域的次数与模型组比较增多(P<0.05);2、致痫后海马尼氏染色可见模型组CA1区神经元内尼氏小体脱失明显,通过存活细胞计数显示各时间点黄芪甲苷可以减轻上述损伤,差异有统计学意义;3、神经胶质纤维蛋白(GFAP)免疫组织化学结果显示:各组海马齿状回区及皮层均可见GFAP免疫反应阳性星形胶质细胞,阳性细胞呈棕褐色,细胞形态如蜘蛛,有突起,突起上有分支,对分布较多的DG区进行观察显示各时间点黄芪甲苷药物干预组较假手术组及癫痫模型组GFAP免疫反应阳性星形胶质细胞减少,差异有统计学意义(p<0.05);4、BrdU免疫组织化学染色研究显示各时间点黄芪甲苷药物干预组较假手术组及癫痫模型组海马齿状回区细胞增殖数目增加(p<0.05),有统计学意义。结论:1.KA导致大鼠出现典型的癫痫样行为学表现和特征性脑电图改变并造成海马神经元变性、死亡,细胞结构瓦解等损伤。充分说明大鼠KA快速点燃模型的建立是成功的。对进一步研究癫痫的发病机制和筛选抗癫痫药物提供了理想的动物模型。2. KA致痫后可造成KA模型大鼠的认知与记忆能力有明显下降。其改变可能与海马神经元变性、死亡,细胞结构瓦解等损伤和星形胶质细胞增生有关。3.黄芪甲苷能减轻癫痫大鼠海马区神经元的死亡,表现为尼氏染色神经元丢失数目明显减少,黄芪干预下KA模型大鼠的认知与记忆能力较模型组有明显改善并能够明显抑制癫痫发生后所引发的星形胶质细胞增生,有明显的神经保护作用。4.黄芪甲苷能诱导癫痫大鼠齿状回区细胞增殖,提示其具有促进神经发生的能力。更多还原

【Abstract】 ojective: Epilepsy is one of the neurodegenerative diseases that is duely and repeatly electric-chemical reaction.As we known,seizures ale associated with a variety of pathophysiological alterations and Can result in neuronal degeneration.On the other hand,neurogenesis could be regulated using external factors such as Epilepsy,PD which results in increased number of neurons.The mechanisms that regulate adult hippocampal neurogenesis have been received major efforts in recent years.As a result,knowledge of the stem cells’microenvironment is fundamental for understanding how a stem cell can proliferate,choose its fate and eventually integrate into mature tissue. Astragaloside IV,the major active structures of Astragalus membranaceus,which has been proved effective inantistress,antioxidation,antiinflammatory,adjust immunity,brain puzzle.We studied the effects of astragalosidesⅣon KA-induced alteration of proliferation in NSC,indicated that the protective effects of astragaloside IV on kA model.Methods: Mature male Sprague-Dawley(SD) rats were separated into three random groups:Sham Operated(SO)group,MODEL group and AST group.Seizures were induced by KA through intracerebroventricular injection by a stereotaxic apparatus. Using introperitioneal injection of 5-bromo-2’-deoxyuridine(BrdU)combined with immunohischemistry for marking proliferating neural cells in the hippocampal dentate gyms,and to observe the dynamic expression of glial fibrilary acidic protein (GFAP)in the development of DG in hippocampal;To evaluate pathology features of hippocampal neurons of every experiment groups,Nissl staining by Toluidine Blue were performed;The cognitive function of the rats was evaluated by Morris water maze test;The research is done for the purpose of observing the function of AST on rat neural stem cells and the effect of AST on the neural stem cells’proliferation,and protect hippocampal neural stem cells,which provides favourable theory knowledge and significant experiment evidences to the prophylaxis and treatment of EP.Results: 1Morris water maze (1)Place navigation test :At all time point on day 1,comparisons of the three groups were of no difference in significance(p>0.05).On day 2,the mean escape latency of rats in model group was much longer than that in the Sham Operated(SO) group (P<0.05), the comparison of ASTgroup and the model group had no distinct difference(P>0.05).During day 3-5,as compared with Sham Operated(SO) group,model and AST groups were significantly prolonged (P<0.05);(2)Spatial probe test:The number of crossing times through the platform quadrant within 120S in model group decreased obviously compared with that in AST group (P<0.01),and the numbers of crossing times through the platform quadrant within 120S in AST group increased significantly compared with that in model group(P<0.05);2Nissle bodies reduced or disappeared in CAl areas in the model groups after SE,and AST could reduce neuron impairment and necrosis on the Hippocampus of epilepsy rats,improve the loss of Nissl bodie(p<0.05);3The results of immunohistochemistry:In all the groups,GFAP-immunoreactive astrocytes can be seen in DG Positive cells were brown with branching processes and like the spiders.At all time point,The expressions of GFAP in DG in the model groups were much higher significantly than that in other groups(P<0.05),Compared with the model groups,expression of GFAP in AST groups decreased significantly (P<0.05);4The results of immunohisto- chemistry:at all time point,Compared with the Sham Operated(SO) group and AST groups,the Brdu+cells in the dentate subgranular cell layer of rats are significantly different(p<0.05).Conclusion: 1 The change of praxiology and pathology of epilepsy type call be expressed ,and electro eneephalogram was detected dynamically by kainic acid epilepsy model.The neuron of hippocampus are obviously death and the cell stracture are destroyed.Therefore,Epilepsy model in rats established by stereotactic iniection of KA can be regarded as one of the ideal model, which is helpful to the study on epilepogenesis,and the antiepileptic activity of the drug.2 Epileptic seizures by KA induced could lead to of diminishing the ability of learning and memory, associate with the neuron death ,the cell stracture and astrocytes proliferation. 3 AST can decrease the hippocampus neuronal death,improve the ability of learning and memory,restraint the hyperplasy of astrocyte in brain.It suggest that AST can protect hippocampal neurons from damage. 4 AST can promote neurogenesis of the endogenous NSCs and neurons ,with a certain degree of neuroprotective effect.更多还原