【作者】 刘超群；

【导师】 何明大；

【作者基本信息】 中南大学 ， 中西医结合临床， 2008， 硕士

【摘要】 目的:观察脑灵汤对阿尔茨海默病模型大鼠行为学、海马组织形态学、海马CA3区GSK-3β和P-CREB蛋白表达的影响,初步探讨脑灵汤对AD模型大鼠的治疗作用及其可能的作用机制。方法:将经Y型迷宫测试筛选后入选的40只SD大鼠完全随机分为正常组、假手术组、模型组、中药组和西药组。采用Aβ1-42注射大鼠海马制作AD大鼠模型,Y迷宫试验和Morris水迷宫测定大鼠造模后三周的学习记忆成绩,取脑组织作冰冻切片,刚果红染色观察大鼠海马是否有β-淀粉样蛋白的沉积,HE染色、Nissl染色观察大鼠海马组织形态学改变。免疫组化法观察GSK-3β和P-CREB在大鼠海马CA3区组织神经元中的表达。结果:(1)Y迷宫实验中,与假手术组和正常组比较,模型组大鼠学会躲避所需的电刺激次数明显增多(p＜0.05),与模型组比较,经药物干预后,中药、西药两治疗组大鼠表现为所需的电刺激次数减少(p＜0.05),且两治疗组相比无显著性差异。(2)Morris水迷宫隐匿平台搜索实验中,与假手术组和正常组比较,模型组大鼠表现为逃避潜伏期明显延长(p＜0.05);探索实验中,正常组和假手术组动物的平均穿越次数最多,中药组和西药组动物穿越次数又多于模型组,比较有显著差异(p＜0.05)。(3)刚果红染色:光镜下,正常组、假手术组大鼠海马切片未见橘红色淀粉样沉积,海马背侧细胞带排列整齐。模型组大鼠Aβ1-42注射部位的海马切片上可见橘红色斑块状淀粉样沉积,提示海马组织区有Aβ沉淀,且模型组海马背侧细胞带明显损伤,局部神经元大段缺失,并出现胶质细胞反应。(4)光镜观察显示,假手术组和正常组大鼠海马CA3区锥体细胞排列紧密整齐,无明显神经元脱失;模型组大鼠海马CA3区锥体细胞排列稀疏、紊乱,神经元脱失明显,且正常锥体细胞数目较正常组和假手术组比较明显减少(p＜0.05);中、西药治疗组大鼠海马CA3区神经元脱失现象明显减轻,锥体细胞形态较正常,排列较整齐,接近假手术组。(5)GSK-3β免疫组化结果显示,各组大鼠海CA3区域GSK-3β阳性细胞数比较,模型组最多,中药组和西药组高于正常组和假手术组(p＜0.05),具有统计学意义。灰度值比较结果显示,模型组显著高于正常组与假手术组,而中药组与西药组低于模型组(P＜0.05),具有统计学意义。(6)P-CREB免疫组化结果显示,各组大鼠海马CA3区P-CREB阳性细胞数比较,模型组最少,中药组和西药组低于正常组和假手术组(p＜0.05),具有统计学意义。各组大鼠海马CA3区P-CREB灰度值比较,模型组显著低于假手术组和正常组(p＜0.05);中药组和西药组大鼠海马CA3区P-CREB灰度值较模型组显著增加(p＜0.05)。结论:(1)本实验研究结果表明采用Aβ1-42注射大鼠海马制作AD模型大鼠出现了AD的一些行为学及病理改变,说明该模型的建立是成功的。(2)AD模型大鼠学习记忆能力明显下降,脑灵汤能提高模型大鼠的学习记忆能力。(3)脑灵汤能减轻AD大鼠海马CA3区神经元脱失现象,改善AD大鼠海马锥体细胞形态,对AD大鼠有治疗作用。(4)脑灵汤可降低AD大鼠海马CA3区GSK-3β的表达,提高P-CREB水平,这些可能是脑灵汤治疗阿尔茨海默病的部分作用机制。更多还原

【Abstract】 Objective:To observe the effect of Naoling Decoction on ethology, hippocamal histomorphology,and expression of GSK-3βand P-CREB in CA3 region of the rats with synthetic Alzheimer disease,and explore the therapeutical effect on Alzheimer disease rats and the potential mechanism of action of Naoling Decoction.Methods:Forty SD(spragu-dawleg)rats were classified into five groups after Y-electric-maze testing:normal group,sham- operated group, model group,Naoling Decoctio group and Piracetam group.The Alzheimer disease model was established by Aβ1—42 injected into hippocamal in rats. The faculty of learning and memory was evaluated by Y-electric-maze test and Morris water maze experiment after establishing AD model.The brain tissues were removed to be made frozen sections and stained with congored to detect the aggradation of Aβ,the changes of cell morphology were detected by HE stain and Nissl stain.Expression of GSK-3β,P-CREB in CA3 region was measured with immunohistochemical staining.RESULTS:(1)The results of Y-electric-maze test showed that compared with sham-operated group and normal group,model group’s times to evade stimulate is more(p＜0.05);compared with model group,Naoling Decoctio group and Piracetam group’s times to evade stimulate is less(p＜0.05). Meanwhile,there existed no obvious difference between these two therapy groups(p＞0.05).(2)The results of Morris water maze experiment showed that Compared with sham-operated group and normal group,the escaped and latent period of model group was delayed significantly(p＜0.05).In exploratory experiment,the average pass through times in sham operation group and normal group were more than that of other three ones,and that of the model group was lest,there were significant difference (p＜0.05).(3)Congored coloration result:Under the light microscope,in the hippocampal fields of rats in sham-operated group and normal group, orange amylaceous aggradation can’t be seen,the cells in DG arrange in order. In the hippocampal fields of rats in the model group,significant orange amylaceous aggradation can be seen in the area that injecting Aβ1—42,this denota there is aggradation of Aβin the hippocampal fields of rats in the model group,meanwhile,the cells in DG obviously damaged、neuron loss noticeable,and there is neuroglial cell proliferation.(4)Under the light microscope,hippocampal CA3 fields of rats in the sham-operated group and normal group exhibited closely and neatly spaced pyramidal cells,and insignificant neuron loss;in the hippocampal CA3 fields of rats in the model group sparse and disturbed pyramidal cells,noticeable neuron loss and neuroglial cell proliferation could be seen,meanwhile,the number of pyramidal cells is less than sham-operated group and normal group(p＜0.05). Neuron loss reduced significantly in the hippocampal CA3 pyramidal cell fields of the two therapy group and cell morphology presented relative normal. (5)The result of GSK-3βwith immunohistochemical staining denote:The compasion of masculine cells about GSK-3βin each group of hippocampal CA3,the model one was the most,the Naoling Decoction group and Piracetam group were more than sham-operated group and normal group(p＜0.05).They had statistical significance.The compasion of the gray scale value about GSK-3βin hippocampal CA3 of each group,the model group were higher than sham-operated group and normal group(p＜0.05);the gray scale value of Naoling Decoction group and Piracetam group were lower than the model group’s gray scale value(p＜0.05).(6)The result of P-CREB with immunohistochemical staining denote:The compasion of masculine cells about P-CREB in hippocampal CA3,of each group,the model one was the least,the Naoling Decoction group and Piracetam group were more than the model group(p＜0.05),They had statistical significance..The compasion of the gray scale value about P-CREB in hippocampal CA3 of each group,the model group were lower than sham-operated group and normal group (p＜0.05);the gray scale value of Naoling Decoction group and Piracetam group were higher than the model group’s gray scale value(p＜0.05).CONCLUTIONS:(1)The presentation of results of the study that the model was established by Aβ1—42 injected into hippocamal was blest, because the model rats had the changes of praxiology and pathology.(2)The learning and memory of the model rats were decreased obviously,Naoling Decoction can improve the learning and memory of the model rats.(3)Naoling Decoction can dcrease neuronal loss and better pathomorphological changes in hippocampal CA3 of AD rats,thusly it has therapeutic action on vascular dementia rats.(4)Naoxintong capsule can weaken the expression of GSK-3βin hippocampal CA3 of AD rats,Elevate the expression of P-CREB,those partly may be therapeutic mechanism on Alzheimer disease of Naoling Decoction.更多还原