脂联素改善三转基因阿尔茨海默病模型小鼠学习记忆能力损伤的神经保护作用及机制研究

发布时间：2018-01-21 13:31

本文关键词： 脂联素三转基因小鼠学习记忆突触可塑性炎症　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨脂联素(Adiponectin,APN)预处理能否有效改善9月龄APP/PS1/tau三转基因阿尔茨海默病(3xTg-AD)模型小鼠的新物体识别、工作记忆和空间学习记忆能力损伤,缓解其焦虑情绪,逆转其在体海马突触可塑性伤害,影响其海马内促炎因子NF-κB,IL-1β、TNF-α及抗炎因子IL-10的表达水平。从而证实脂联素在AD病程中是否能发挥神经保护作用,并探讨其机制是否与减轻神经炎性反应有关。方法:实验选用9月龄雄性3xTg-AD小鼠和其背景鼠C57BL/6J小鼠,随机分为四组,每组8只,分别为:野生对照组(WT+Saline)、野生给药组(WT+APN)、三转模型组(3xTg+Saline)和三转给药组(3xTg+APN)。连续10天经侧脑室分别注射0.1μg/4μl的脂联素或等体积的生理盐水,然后开始行为学实验,在实验期间持续给药直至断头取海马组织。行为学实验分为四部分依次进行:(1)旷场实验:在安静环境下,把小鼠放入旷场中央自由探索5 min,记录小鼠运动的总距离及其在中央区域或外周区域的活动时间占总时间的百分比。(2)新物体识别实验:旷场实验后第2天进行。在熟悉实验阶段,将两个完全相同的物体放入旷场,再将小鼠从两物体对侧壁的中点贴壁放入,让其自由探索10 min。小鼠休息5 h后,进行测试实验,将其中一个物体换为材质相同、颜色形状不同的新物体,让小鼠再自由探索10 min。分析指标包括物体识别指数和辨别指数,物体识别指数=探索新物体时间或探索旧物体时间/(探索新物体时间+探索旧物体时间)×100%,辨别指数=(探索新物体的时间-探索旧物体的时间)/(探索新物体时间+探索旧物体时间)×100%。(3)Y迷宫自发交替实验:将小鼠放入Y迷宫的三臂交汇区自由活动8 min,记录小鼠进入各臂的总次数和顺序,并计算其自发交替正确率。(4)Morris水迷宫实验:首先进行5天的定位航行实验,每天将小鼠随机放入4个象限。记录其游泳轨迹、游泳速度和逃避潜伏期(找到水下平台所用时间)。第6天上午进行空间探索实验,记录60 s内小鼠的运动轨迹及在目标象限的活动时间;下午进行可视平台实验,记录小鼠找到平台的时间和游泳速度。行为学实验结束后,进行在体海马场电位记录实验。将小鼠麻醉后固定在脑立体定位仪上,定位其海马CA1区并将绑定的刺激电极和记录电极插入。首先记录30 min场兴奋性突触后电位(field excitatory postsynaptic potentials,f EPSPs),然后给予配对刺激记录双脉冲易化(paired-pulse facilitation,PPF),最后通过高频刺激(high-frequency stimulation,HFS)诱导长时程增强(long term potentiation,LTP)并记录60 min。通过比较HFS前后各组f EPSPs的斜率来反映AD相关基因突变和脂联素预处理是否影响突触可塑性;通过比较各组的PPF值,来确定是否有突触前神经递质释放的改变。在体LTP实验后,将小鼠断头取脑,通过Western blot实验检测各组小鼠海马内促炎因子NF-κB、IL-1β、TNF-α和抗炎因子IL-10的表达量。结果:(1)脂联素可以明显改善9月龄3xTg-AD小鼠的识别记忆、工作记忆和空间学习记忆能力损伤,并缓解其焦虑情绪。在旷场实验中,与WT+Saline组相比,3xTg+Saline组小鼠在中央区域活动的时间百分比明显降低(P0.001),而在外周区域活动的时间百分比明显升高(P0.001),表明3xTg小鼠出现焦虑情绪;给予APN后,3xTg小鼠在中央区域活动的时间增加,而在外周活动的时间减少(P0.01),其焦虑情绪得到缓解。在新物体识别实验中,除3xTg+Saline组小鼠的新旧物体识别指数之间没有统计学差异外,其余各组小鼠的新物体识别指数明均显高于旧物体识别指数(P0.05)。同时,3xTg+Saline组小鼠的辨别指数明显低于WT+Saline组(P0.01),APN预处理后提高了3xTg小鼠的辨别指数(P0.01),表明APN能够有效改善3xTg小鼠的新物体识别记忆损伤。在Y迷宫实验中,各组小鼠的总进臂次数没有统计学差异,但3xTg+Saline组小鼠的自发交替正确率明显低于WT+Saline组和3xTg+APN小鼠(P0.01),提示脂联素能够改善3xTg小鼠的工作记忆损伤。在Morris水迷宫定位航行实验中,各组小鼠的逃避潜伏期均随训练天数增加而明显缩短。在第2-5天,3xTg+Saline组小鼠找到平台所需时间明显长于WT+Saline组(P0.05);在第3-5天,3xTg+APN组小鼠的逃避潜伏期明显短于3xTg+Saline组小鼠(P0.05)。在空间探索实验中,3xTg+Saline组小鼠在目标象限的活动时间百分比明显少于其余三组(P0.001),且各组小鼠在6天中的游泳速度和到达可视平台的时间均无统计学差异(P0.05),表明脂联素预处理改善了3xTg小鼠空间学习记忆能力损伤。(2)脂联素逆转9月龄3xTg-AD小鼠的海马突触可塑性伤害。各组小鼠在HFS前30 min所记录的f EPSPs斜率和配对脉冲刺激所引起的PPF比值均没有统计学差异(P0.05)。当给予HFS后,在四组小鼠均成功诱导出LTP。但在HFS后20 min开始,3xTg+Saline组小鼠f EPSPs的斜率明显降低,在HFS后30 min和60 min,3xTg+Saline组小鼠f EPSPs的斜率都明显低于WT+Saline组和3xTg+APN组小鼠(P0.05)。以上结果表明,AD相关基因突变和脂联素预处理均未影响基础突触传递和突触前神经递质释放,但脂联素预处理可以有效逆转3xTg小鼠的海马突触可塑性损伤。(3)脂联素减轻了3xTg-AD小鼠海马内的神经炎性反应。Western blot实验结果显示:与WT+Saline组相比,3xTg+Saline组小鼠海马内促炎因子NF-κB、IL-1β和TNF-α的表达量明显升高(P0.01),而抗炎因子IL-10的表达量却明显降低(P0.001)。给予APN后,3xTg-AD小鼠海马内NF-κB、IL-1β和TNF-α的表达量有所下降(P0.05),而IL-10的表达量有所升高(P0.01)。结论:9月龄3xTg-AD小鼠的识别记忆、短期工作记忆和长期空间学习记忆能力明显损伤,并出现焦虑情绪。同时,3xTg-AD小鼠还表现出在体海马LTP的抑制和海马内神经炎性反应。给予脂联素后,能够改善3xTg-AD小鼠的学习记忆能力损伤、缓解其焦虑情绪和逆转海马LTP抑制,并且脂联素可能是通过减少促炎因子表达和增加抗炎因子分泌,减轻脑内神经炎性反应,在AD病程中发挥神经保护作用的。
[Abstract]:Objective: To investigate the effect of adiponectin (Adiponectin, APN) pretreatment can effectively improve the September age three APP/PS1/tau transgenic Alzheimer's disease (3xTg-AD) mouse model of new object recognition, working memory and spatial learning and memory damage, alleviate their anxiety to reverse the in vivo hippocampal synaptic plasticity in the hippocampus damage effects of proinflammatory cytokines NF- K B, IL-1 beta, the expression level of TNF- alpha and anti-inflammatory factor IL-10. To verify whether adiponectin in the course of AD can exert neuroprotective effects, and to explore its mechanism is associated with reduced neuroinflammatory response. Methods: twenty September old male 3xTg-AD mice and its background in C57BL/6J mice were randomly divided into four groups, each group 8, respectively: control group (WT+Saline), wild wild drug group (WT+APN), three (3xTg+Saline) and model group (3xTg+APN group) to three. 10 consecutive days were injected into lateral ventricle of 0.1 g/4 L The volume of saline or adiponectin, and behavioral experiments, during the experiment continuous administration until decapitated in hippocampus. The behavior experiment is divided into four parts: (1) in the open field test: in a quiet environment, the mice in the open field of central free exploration of 5 min, the total distance of movement of mice record and in the central region or peripheral regions of the percentage of the time in total time. (2) a new object recognition experiment: in the open field second days after the experiment. With the experimental stage, the two same objects in the open field, and then the mice from the two object point to the side wall of the wall let the freedom to explore into 10 min. mice rest after 5 h test, will be one of the objects for the same material, the new color of objects of different shapes, so the mice were then free to explore the 10 min. indicators including index index and object recognition to identify objects To explore a new object recognition index = time or time / explore old objects (Exploring the novel object exploration of old time + time object) * 100%, index of discrimination (= time exploring the novel object exploration of old objects (time) / time to explore new objects and explore the old object time) * 100%. (3) Y spontaneous maze alternate experiment: the mice were placed in the three arm maze of Y free intersection area of 8 min, and the total number of mice in each arm of the order of record, and calculate the correct rate of spontaneous alternation. (4) Morris water maze test: first locate 5 days of sailing experiment, mice with daily machine into the 4 quadrants. The swimming trajectory, swimming speed and escape latency (the time to find the platform under water). The morning of the sixth day to carry out space exploration experiment, recorded within 60 s mice and trajectories in the target quadrant time; the afternoon visual experimental platform, the platform to find records of mice And swimming speed behavior. After the experiment, the sea horse potential recording experiment. The mice were anesthetized on the stereotaxic apparatus, the location of the CA1 region of the hippocampus and the stimulating and recording electrodes inserted into the binding. First recorded in 30 min field excitatory postsynaptic potential (field excitatory postsynaptic potentials. F EPSPs), and then given the paired stimuli recorded paired pulse facilitation (paired-pulse facilitation, PPF), and finally by high frequency stimulation (high-frequency stimulation, HFS) induced long-term potentiation (long term, potentiation, LTP) and recorded 60 min. by comparing the slope of HFS f before and after EPSPs to reflect the AD gene mutation and adiponectin pretreatment whether the treatment effect of synaptic plasticity; through the comparison of the PPF value, to determine whether the presynaptic neurotransmitter release in the body. The change of LTP after the experiment, the mice brains were removed by W. Detection of estern blot mice hippocampus in experimental inflammatory factor NF- kappa B, IL-1 beta, TNF- alpha and the expression of anti-inflammatory factor IL-10. Results: (1) adiponectin can significantly improve the September age 3xTg-AD mice recognition memory, working memory and spatial learning and memory deficits, and alleviate their anxiety in the open field test. Compared with the WT+Saline group, 3xTg+Saline group, mice in the central area of activity time was decreased (P0.001), and the percentage of time in the peripheral areas of activity increased significantly (P0.001), showed that 3xTg mice had anxiety; for APN, the time in the central areas of activity of 3xTg mice increased in the peripheral the less time (P0.01), the anxiety eased. In the new object recognition experiments, in addition to no statistically significant difference between the old and the new object recognition index in 3xTg+Saline group, the new object recognition refers to the remaining mice The numbers were significantly higher than that of the old object recognition index (P0.05). At the same time, the 3xTg+Saline group discrimination index was significantly lower than in group WT+Saline (P0.01), APN after pretreatment improves the discrimination index of 3xTg mice (P0.01), show that APN can effectively improve the new object recognition memory in 3xTg mice in Y maze injury., no statistically significant differences between the total number of mice into the arm, but the spontaneous alternation of mice in 3xTg+Saline group was significantly lower than that of WT+Saline group and the correct rate of 3xTg+APN mice (P0.01), suggesting that adiponectin can improve working memory impairment in 3xTg mice. In Morris water maze navigation experiment, mice escape latency was increased significantly with the number of training days shortened. On day 2-5, 3xTg+Saline group of mice to find the platform required time was longer than that of group WT+Saline (P0.05); in the first 3-5 days, 3xTg+APN group mice escape latency was significantly shorter in the 3xTg+Saline group Rat (P0.05). In the space exploration experiment, 3xTg+Saline group in the target quadrant time percentage was significantly less than the other three groups (P0.001), and the swimming speed in 6 days in mice and reached the visual platform of the time were not statistically significant (P0.05), showed that adiponectin pretreatment improved the space 3xTg learning and memory ability of mice damage. (2) injury of hippocampal synaptic plasticity of adiponectin reversed in September aged 3xTg-AD mice. PPF mice in the ratio of 30 before HFS min recorded f EPSPs slope and paired pulse stimulation had no significant difference (P0.05). When given after HFS, in four groups of mice were successfully but after HFS induced LTP. 20 min, f EPSPs slope 3xTg+Saline group were significantly decreased after HFS, 30 min and 60 min, the slope of F EPSPs in 3xTg+Saline group were significantly lower than those of group WT+Saline and group 3xTg+APN mice (P0.05). The above results The result showed that the AD gene mutation and adiponectin pretreatment did not affect basal synaptic transmission and presynaptic neurotransmitter release, but adiponectin pretreatment can effectively reverse the 3xTg mice hippocampal synaptic plasticity damage. (3) adiponectin reduced 3xTg-AD mouse hippocampus in neuritic reaction of.Western blot results showed that compared with the WT+Saline group 3xTg+Saline group of proinflammatory cytokines in mice hippocampus NF- kappa B, expression of IL-1 beta and TNF- alpha were significantly increased (P0.01), whereas the expression of anti-inflammatory factor IL-10 was significantly lower (P0.001). After administration of APN, 3xTg-AD in NF- mice hippocampus kappa B, expression of IL-1 beta and TNF- alpha declined (P0.05), whereas the expression of IL-10 increased (P0.01). Conclusion: September 3xTg-AD mice at the age of recognition memory, short-term working memory and long-term spatial learning and memory ability was damaged and anxiety. At the same time, but also the performance of 3xTg-AD mice A neuroinflammatory response in the hippocampus and inhibition of hippocampal LTP. Given adiponectin, the ability of learning and memory damage can improve 3xTg-AD mice, alleviate their anxiety and reverse the inhibition of hippocampal LTP, and adiponectin may be reduced by proinflammatory cytokine expression and increased inflammatory factor secretion, reduce inflammatory reaction in brain in AD in the course of neuroprotection.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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