缺锌致大鼠学习记忆损伤的信号转导机制研究

发布时间：2018-01-06 21:16

  本文关键词：缺锌致大鼠学习记忆损伤的信号转导机制研究　出处：《中国人民解放军军事医学科学院》2008年硕士论文　论文类型：学位论文

  更多相关文章： 缺锌 海马 信号转导 学习记忆 CREB

【摘要】： 目的:在成功制备缺锌大鼠动物模型的基础上,观察缺锌对生长大鼠认知行为、突触传递效能、抗氧化功能及海马神经细胞凋亡的影响,重点探讨缺锌对生长期大鼠海马cAMP/PKA和MEK/ERK信号通路中几种重要信号分子表达的影响,以期阐明缺锌致认知功能损伤的信号转导机制。方法:(1)缺锌大鼠模型的建立。健康雄性Wistar大鼠96只,体重60g～80g,随机分为三组:足锌组(ZA)、缺锌组(ZD)、对喂组(PF),其中ZD组喂饲缺锌饲料(1.5mg/kg),ZA组和PF组喂饲足锌饲料(30mg/kg)。以当日ZD组摄食量作为次日PF组的给料量。动物单笼喂养,自由饮用去离子水。实验期一个月。(2)锌营养状况评价。采用火焰原子吸收法测定大鼠血浆锌和海马锌含量,TSQ荧光法检测海马CA3区锌的分布和含量,连续监测法测定血浆碱性磷酸酶(ALP)活性。(3)学习记忆能力检测。通过被动回避实验和Y-迷宫实验观察缺锌对实验大鼠学习记忆能力的影响。(4)LTP诱导实验。通过诱导大鼠海马齿状回LTP观察缺锌对大鼠海马突触传递效能的影响。(5)生化指标的检测。采用碱性羟胺比色法分别测定全血、海马及皮层乙酰胆碱(Ach)的含量;荧光分光光度法测定海马、皮层单胺类神经递质多巴胺(DA)、去甲肾上腺素(NE)、5-羟色胺(5-HT)的含量。采用硫代巴比妥酸法测定大鼠血清、海马和皮层丙二醛(MDA)含量;采用相关试剂盒分别检测大鼠血清、海马和皮层超氧化物歧化酶(SOD)活性、总抗氧化力(T-AOC)。以流式细胞术检测大鼠海马神经细胞凋亡率。(6)信号通路中信号分子的检测。采用放射免疫分析法(RIA)测定大鼠血浆、海马和皮层组织cAMP含量;应用PepTag?检测系统测定海马和皮层PKA活性。分别应用Western blot和RT-PCR法检测大鼠海马和皮层p-MEK、p-ERK1/2、p-CREB和BDNF蛋白的表达水平以及CREB mRNA、BDNF mRNA的转录水平。结果:(1)实验期间,ZD组大鼠精神萎靡,活动减少,生长迟缓,并逐渐出现明显的脱毛、嘴角炎和趾间炎等缺锌症状。(2)与ZA组和PF组比较,ZD组大鼠体重、摄食量、血浆锌含量、海马CA3区锌含量、ALP活力均明显降低(P0.05)。(3)与ZA组和PF组相比,ZD组大鼠的避暗潜伏期明显缩短(P0.05),在Y迷宫测试中的错误反应次数显著增加(P0.05)。(4)三组大鼠均诱导出LTP;但ZD组大鼠在高频刺激后,各个时间点的群峰电位(PS)增长幅度较ZA组和PF组显著降低(P0.05)。(5)与ZA组和PF组相比,ZD组大鼠全血Ach含量显著升高(P0.05),海马Ach含量有下降趋势,但无显著性差异;ZD组大鼠皮层Ach含量与ZA组相比显著降低(P0.05),但与PF组比较无显著性差异;ZD组脑组织NE、5-HT、DA等单胺类神经递质含量较ZA组和PF组不同程度升高(P0.05)。(6)与ZA组和PF组相比,ZD组大鼠血清、海马和皮层MDA含量显著增加(P0.05), SOD活性及T-AOC降低(P0.05);并且ZD组大鼠海马神经细胞凋亡率显著升高(P0.05)。(7)与ZA组比较,ZD组和PF组大鼠海马cAMP含量显著升高(P0.05),ZD组较PF组有升高趋势;而ZD组大鼠血浆和皮层cAMP含量显著高于ZA组和PF组(P0.05),ZA组与PF组间无差异;ZD组大鼠海马和皮层PKA活性较ZA组和PF组均显著降低(P0.05),PF组又显著低于ZA组(P0.05)。(8)ZD组海马和皮层p-MEK、p-ERK1/2蛋白表达水平较ZA组和PF组均明显下调,而ZA组和PF组之间没有显著性差异。(9)与ZA组和PF组相比, ZD组海马和皮层p-CREB、BDNF蛋白表达水平及CREB mRNA、BDNF mRNA水平均明显下调,而ZA组和PF组之间也分别有不同程度的改变。结论:(1)成功建立了缺锌大鼠动物模型。(2)锌缺乏可使大鼠学习记忆功能受损。(3)锌缺乏可导致大鼠海马脑区突触传递效能降低,胆碱能及单胺类神经递质含量异常改变,抗氧化防御系统功能降低,海马神经元凋亡率增高,进而在行为上表现出学习记忆功能降低。(4)锌缺乏可导致大鼠海马和皮层cAMP-PKA及MEK-ERK信号通路中主要信号分子发生异常改变,使CREB磷酸化水平下降,BDNF蛋白表达水平下降,从而抑制LTP的形成并损伤学习记忆,这可能是缺锌致学习记忆功能损伤的重要分子机制。
[Abstract]:Objective: to prepare basic zinc deficient rats in animal models were successfully on the observation of zinc deficiency on the growth of cognitive behavior in rats, synaptic transmission efficiency, the effects of antioxidant function and neuronal apoptosis in the hippocampus, focusing on the effects of zinc deficiency on the expression of several important signal molecules in rat hippocampal cAMP/PKA and MEK/ERK signaling pathway in the growth period, in order to clarify the signal transduction mechanism of zinc deficiency induced cognitive impairment. Methods: (1) establishment of zinc deficient rat model. 96 healthy male Wistar rats, weight 60g ~ 80g, were randomly divided into three groups: foot group (ZA), zinc deficiency group (ZD), pair fed group (PF), ZD group fed with zinc deficient diet (1.5mg/kg), ZA group and PF group were fed the zinc adequate feed (30mg/kg). On the same day as the food intake of group ZD group PF the next day to feed the animal. Single cage feeding, drinking deionized water. The experimental period of one month. (2) evaluate the zinc nutritional status was determined by large. Flame atomic absorption spectrometry Plasma zinc and zinc content in hippocampus, the content and distribution of detection in hippocampal CA3 region of zinc TSQ fluorescence method for determination of plasma alkaline phosphatase (ALP) activity of continuous monitoring method. (3) the ability of learning and memory testing. Through the passive avoidance test and Y- maze test to evaluate the influence of zinc deficiency on rat memory ability of learning (4.) induced by LTP experiment. Through the induction of LTP in the rat dentate gyrus examined effects on hippocampal synaptic transmission efficacy in rats. (5) biochemical detection. Blood were measured by colorimetric method of alkaline hydroxylamine, hippocampus and cerebral cortex acetylcholine (Ach) content of hippocampus; determination of fluorescence spectrophotometry, cortex monoamine the neurotransmitter dopamine (DA), norepinephrine (NE), 5- serotonin (5-HT). The contents of serum were measured by thiobarbituric acid method, the hippocampus and the cortex malondialdehyde (MDA) content in serum of rats were detected; the related kit, hippocampus and skin The layer of superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC). To detect the apoptosis rate of hippocampal nerve cells by flow cytometry. (6) the detection signals in signalingpathways. Radioimmunoassay (RIA) determination of rat plasma, the content of cAMP in hippocampus and cortex tissue; application PepTag? PKA activity in hippocampus and cortex were detected respectively by the Western system. Blot and RT-PCR were detected in the cortex and hippocampus of p-MEK, p-ERK1/2, p-CREB and BDNF protein expression levels of CREB and mRNA, the transcriptional level of BDNF mRNA. Results: (1) during the experiment, ZD rats, listlessness, decreased activity, growth slowly and gradually apparent hair removal, and toe inflammation mouth inflammation and other symptoms of zinc deficiency. (2) compared with ZA group and PF group, ZD group, body weight, food intake, plasma zinc content, zinc content of CA3 in hippocampus, the activity of ALP was significantly decreased (P0.05) compared with ZA (3). Group and PF group, ZD group 榧犵殑閬挎殫娼滀紡鏈熸槑鏄剧缉鐭，

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