淀粉样β蛋白所致大鼠突触素及其行为学改变

Changes of synaptophysin and behavior in rats induced by beta-amyloid protein

目的:观察淀粉样β蛋白对大鼠学习记忆能力及突触含量的影响。方法:实验于2004-02在郑州大学医学院生理学教研室完成。实验动物为三四月龄的健康雄性SD大鼠(Y迷宫筛选对电击敏感并逃避迅速)24只,随机数字表法分成正常对照组、假手术组、阿尔茨海默病模型组,每组8只,大鼠双侧海马微量注射聚合态淀粉样β蛋白1-40制备阿尔茨海默病动物模型,Y-型迷宫测定大鼠学习记忆能力。①学习测试:规定大鼠受电击后从起步区直接逃至安全区为正确反应,以连续10次中有9次(9/10)正确反应前所需的电击次数(即尝试次数)表示其学习获得能力。若尝试次数超过100次则不再测试,并以100次为最大值计数。以正确反应数占总测试数的百分比计算正确反应率。②记忆再现测试:大鼠休息24h后,同法检测其记忆力。以达9/10标准前的尝试次数表示记忆再现能力,并计算其正确反应率。随后处死大鼠,行突触素免疫组织化学染色。选择海马CA1区利用捷达801系列形态分析系统测定突触素免疫产物的平均吸光度,并以其代表突触素的量。每例标本检测4张切片,求其平均值,再计算每组动物的均值。结果:各组大鼠无意外死亡,全部结果进入结果分析。①光镜下观察突触素的产物呈颗粒状,主要分布在海马CA1、CA3区多形层和分子层及齿状回,呈点状沿多形层和分子层长轴分布。阿尔茨海默病模型组海马CA1区多形层和分子层突触素染色较正常对照组浅。②假手术组大鼠海马结构内突触素吸光度值与对照组接近,差异无显著性意义[(0.268±0.01),(0.270±0.01),t=1.549,P>0.05];阿尔茨海默病模型组大鼠海马结构内突触素吸光度值明显低于对照组,差异有显著性意义[(0.176±0.01),(0.270±0.01),t=2.875,P<0.05]。③阿尔茨海默病模型组大鼠学习能力和记忆能力均显著下降,学习记忆获得尝试次数显著高于正常组和假手术组(P<0.05),并且在整个过程中正确反应率明显低于正常组和假手术组(P<0.05)。结论:海马内注射淀粉样β蛋白1-40可引起大鼠学习记忆能力下降,淀粉样β蛋白1-40所致阿尔茨海默病模型大鼠学习记忆减退与海马CA1区突触的减少有关。

AIM： To study the effects of amyloid-β protein on learning and memory abilities as well as synaptophysin content in rats. METHODS： The experiment was conducted in the Department of Physiology, Medical College of Zhengzhou University in February 2004. Twenty-four experimental animals, which were healthy male SD rats and 3-4 months old （sensitive to shock and rapid in escaping in the Y maze screening） were used and randomly divided into normal control group, sham-operation group and Alzheimer disease group with 8 rats in each group. Polymerized amyloid-β protein 1-40 was microinjected into the bilateral hippocampus of rats to establish model of Alzheimer disease. Y- maze was used to evaluate the learning and memory abilities of rats.①Learning test： correct reaction was regulated as rats run away from the first step area to the safe area after shock. Shock times （test times） of correct reaction in 9 times out of 10 continuous times was taken as the obtaining ability of learning. Rats with test times greater than 100 would not be tested, and 100 times was taken as the maximum. The rate of correct reaction was obtained by calculating the percentage of correct-reaction times in total test times.②Memory reappearance test： Rats were tested of the memory ability after resting for 24 hours. The memory reappearance ability was represented by the trying times below 9/10 of the standard, and the rate of correct reaction was calculated. Rats were then executed to conduct synaptophysin immunohistochemical stain. The morphous analytical system of Jieda 810 series was used to determine the average absorptance of immune products of synaptophysin in hippocampla CA1, which represented the amount of synaptophysin. Four sections in each sample was detected to obtain the mean value, and the mean value of animals in all groups were calculated. RESULTS： All mice were involved in the analysis of results, and no one died.①The products of synaptophysin were in granules under light microscope, which were mainly distributing in polymorphic layer, molecular layer and dentate fascia of CA1 and CA2 in hippecampus. The products distributed along the long axis of polymorphic layer and molecular layer and were in punctiform. The stain of synaptophysin in polymorpic layer and molecular layer of CAI region was lighter in the Alzheimer disease model group than normal control group.②The absorptances of synaptophysin in hippocampus of rats in sham-operation group and control group were similar, and the differences were not significant [（0.268±0.01）, （0.270±0.01）, t=1.549, P 〉 0.05], while that in the A.lzheimer disease model group was significantly lower than the control group with remarkable differences [（0.176±0.01）, （0.270±0.01） ,t=2.875, P 〈 0.05].③The abilities of learning and memory of rats in the Alzheimer disease model group obviously worsened, and the trying times of learning and memory was markedly higher than normal control group and sham-operation group （P 〈 0.05）, moreover, the rate of correct reaction in the whole process was significantly lower than normal control group and sham-operation group （P 〈 0.05）. CONCLUSION： Injection of amyloid-β protein 1-40 in the hippocampus can induce the descending of learning and memory abilities. The failure of memory in rat model with Alzheimer disease induced by amyloid-β protein 1-40 may relate with the decrease of synaptophysin in CA1 of hippocampus.