淀粉样前体蛋白可溶性α片段与睡眠的相关性及潜在的认知保护作用

发布时间：2018-03-10 11:31

本文选题：淀粉样前体蛋白可溶性α片段　切入点：睡眠-觉醒周期　出处：《第二军医大学》2012年博士论文　论文类型：学位论文

【摘要】：【目的】淀粉样前体蛋白（APP）经淀粉样代谢途径产生的可溶性β淀粉样蛋白（Aβ）不正常的聚集与阿尔茨海默病（AD）密切相关。睡眠剥夺影响认知功能，长期睡眠限制的APP转基因鼠可以出现病理Aβ沉积，提示睡眠缺失有可能通过影响APP代谢途径参与AD的致病过程。生理条件下，APP主要经非淀粉样代谢途径产生可溶性α片段（sAPPα）并发挥生理保护作用。在近年多领域针对APP淀粉样代谢途径临床干预研究未获得预期效果的背景下，重新审视和研究APP非淀粉样代谢途径产物sAPPα是否存在与睡眠相关的变化以及在睡眠剥夺动物模型中是否发挥认知保护机制具有一定现实意义。观察sAPPα在基于睡眠改善的临床干预研究中的变化及相关认知能力改变，有可能为认知障碍疾病探寻新的干预或治疗靶点。【方法】建立正常睡眠-觉醒模型和睡眠剥夺模型，观察实验动物是否存在与睡眠相关的sAPPα变化，以及sAPPα是否能够对抗睡眠剥夺引起的认知功能损害；进而观察慢性失眠患者和AD患者药物干预睡眠缺失前后的sAPPα变化以及相应的认知功能评分改变。所有涉及本课题的啮齿类动物在模拟外界光暗变化（12h:12h）的条件下适应性饲养2周，建立正常的睡眠-觉醒周期。首先，利用寰枕膜穿刺技术在24h内按时间节点（间隔4h）获取正常睡眠-觉醒周期Sprague-Dawley（SD）大鼠脑脊液（CSF）样本，利用免疫印迹技术（western-blot）检测脑脊液sAPPα的水平，判断其是否存在昼夜节律性变化。其次，给予上述实验动物12h睡眠剥夺以及12h恢复性睡眠，获取此两节点CSF样本，同样利用免疫印迹技术观察急性睡眠剥夺与睡眠恢复对sAPPα的影响。此外，利用同上的方法观察亚急性部分性睡眠剥夺C57BL6小鼠脑内sAPPα的影响。第三，观察SD大鼠急性睡眠剥夺对认知水平（Morris水迷宫空间记忆）的影响，以及脑内皮质和海马sAPPα的变化，进而观察脑内注射重组sAPPα是否可以对抗睡眠剥夺对Morris水迷宫空间记忆的影响。第四，通过症状询问、查体、量表评估等手段完成AD患者受试对象和慢性失眠患者受试对象的纳入工作，匹配健康对照组。针对各组进行认知量表评定、夜间多导睡眠监测（PSG），并在知情同意后行腰椎穿刺获取脑脊液标本。分析各组认知评分、PSG监测数据以及脑脊液sAPPα之间的差异，给予右佐匹克隆（3mg/d，2周）治疗AD患者和慢性失眠患者的失眠症状，观察治疗前后失眠缓解的情况以及相应的认知评分和脑脊液sAPPα变化。【结果】本研究的结果显示（1）SD大鼠24h内自然睡眠-觉醒周期中的6个时间节点脑脊液sAPPα水平发生具有统计学意义的波动性变化（p=0.013）。其中，sAPPα水平于睡眠始发约4h后达到高峰，而后逐渐下降，觉醒时维持相对稳定的水平。（2）SD大鼠在急性完全睡眠剥夺12h后脑脊液中sAPPα水平下降，在恢复睡眠12h后上升（p=0.024），与环境对照之间具有显著性区别（p=0.011）。（3）与正常对照组和环境对照组相比，C57BL6小鼠在经历亚急性部分性睡眠剥夺2周后脑组织中无论是皮质还是海马sAPPα水平均有统计学意义的下降（p0.05），但皮质与海马之间sAPPα的浓度无统计学意义上的差异（p0.05）。（4）睡眠剥夺可以影响SD大鼠Morris水迷宫空间记忆（目标象限游泳时间百分比）能力（p=0.000），脑室注射重组sAPPα可以改善空间记忆的损害（p=0.037），这种改善在2次注射后更加显著（p=0.005）。（5）与健康对照相比，AD患者和慢性失眠患者均有睡眠效率减低（p=0.000），睡眠期间觉醒增加（p=0.000）以及REM睡眠减少（p=0.000）；AD患者觉醒时间较慢性失眠患者增加（p=0.018），，而慢性失眠患者睡眠潜伏期明显延长（p=0.000）。（6）AD患者脑脊液sAPPα水平较慢性失眠患者和健康对照下降（p=0.000，p=0.000），后两者之间无显著差异（p0.05）。（7）AD患者脑脊液sAPPα水平与认知量表评分和睡眠效率之间具有显著的正相关（spearman’s rho0.676, p=0.016;0.935, p=0.000）。（8）右佐匹克隆干预治疗失眠可以明显改善PSG睡眠参数（p0.05）。（9）右佐匹克隆干预可以改善慢性失眠患者认知量表评分（p=0.047），但不改变脑脊液sAPPα水平（p=0.142）；干预治疗可以增加AD患者脑脊液sAPPα水平（p=0.004），但AD患者认知量表评分没有显著改善（p=0.514）。【结论】首先，sAPPα在正常睡眠-觉醒状态下呈节律性变化，在睡眠期达到高峰。无论是急性完全睡眠剥夺还是亚急性部分性睡眠剥夺均可导致脑内sAPPα水平的下降，急性睡眠剥夺导致的下降在恢复性睡眠之后可以迅速恢复，提示睡眠与脑内sAPPα水平具有相关性。其次，脑内注射重组sAPPα可以对抗急性完全睡眠剥夺动物模型中空间记忆的损害。第三，AD患者脑脊液中sAPPα水平下降，与认知评分及睡眠时间呈正相关，同样存在失眠症状的慢性失眠患者sAPPα水平正常。药物干预改善失眠症状后，AD患者脑脊液sAPPα水平增加，但认知评分无显著恢复，而针对失眠的干预治疗可以改善慢性失眠患者的认知评分。说明人体中脑内sAPPα水平与睡眠存在相关性，改善睡眠可以增加脑内sAPPα水平，但是对于已经发展成阿尔茨海默病的患者，睡眠的改善不能逆转认知功能缺损。
[Abstract]:[Objective] the amyloid precursor protein (APP) soluble amyloid beta protein produced by amyloid metabolic pathways (A beta) aggregation and Blzheimer's disease is not normal (AD) are closely related. The effects of sleep deprivation on cognitive function, long-term sleep restriction of APP transgenic mice can appear pathological A deposition, suggesting that lack of sleep may the pathogenic effects of APP metabolic pathway in AD. Under physiological conditions, APP mainly by non amyloid metabolic pathways to produce soluble fragment of alpha (sAPP alpha) and its physiological protective effect. In recent years many fields in APP amyloid metabolic pathways of clinical intervention did not achieve the expected effect of the background, review and study of APP non amyloid metabolism product of sAPP alpha if there are changes related to sleep and sleep deprivation in animal models of cognitive play protection mechanism has a certain practical significance. Based on the observation of sAPP alpha Changes in the clinical intervention study of sleep improvement and related cognitive changes may be possible to explore new interventions or targets for cognitive impairment.
[Methods] to establish normal sleep wake model and sleep deprivation model, observe the experimental animal existence of sAPP alpha changes and sleep related, and whether the sAPP alpha against cognitive impairment caused by sleep deprivation; and observation of patients with chronic insomnia and drug intervention in patients with AD sAPP alpha changes before and after sleep deprivation and the corresponding cognitive function score change. All refer to this paper in rodent animal simulation of external light dark changes (12h:12h) under the condition of adaptive feeding for 2 weeks, the establishment of normal sleep wake cycle. First of all, the atlanto occipital membrane puncture technique according to the time node within 24h (4h interval) to get normal sleep wake cycle Sprague-Dawley (SD) rat cerebrospinal fluid (CSF) samples by Western blot (Western-blot) levels in cerebrospinal fluid of sAPP alpha, to determine whether it has circadian change. Secondly, given the above experiment Animal 12h sleep deprivation and 12h recovery sleep, get the two node CSF samples, the same observation of acute sleep deprivation and recovery effects on sAPP alpha sleep by Western blot. In addition, the observation of subacute part of the impact of sleep deprivation in C57BL6 mice brain using sAPP alpha ibid method. Third, observation of acute sleep SD after deprivation on cognitive level (Morris water maze memory space) effects, and changes in the brain cortex and hippocampus of sAPP alpha, and observe whether intracerebral injection of recombinant alpha sAPP can counteract the effects of sleep deprivation on Morris water maze spatial memory. Fourth, inquiry, examination by symptoms scale assessment method AD subjects and patients with chronic insomnia subjects included in the work, the healthy control group. The cognitive assessment scales for each group, nocturnal polysomnography (PSG), and informed consent was waist Lumbar puncture CSF samples obtained were analyzed. The differences between cognitive scores, PSG monitoring data and cerebrospinal fluid of sAPP alpha, given dexzopiclon (2 weeks 3mg/d) treatment of patients with AD and patients with chronic insomnia insomnia symptoms were observed before and after treatment of insomnia relief and the corresponding cognitive score and cerebrospinal fluid sAPP alpha changes.
[result] the results of this study show that (1) natural sleep SD rats 24h awakening cycle in 6 time nodes of cerebrospinal fluid sAPP levels fluctuate change was statistically significant (p=0.013). The levels of sAPP in sleep onset after about 4H reached the peak, then decreased gradually, to maintain a relatively stable the level of awakening. (2) sAPP in cerebrospinal fluid of acute 12h after total sleep deprivation in the alpha level of SD rats decreased, increased in sleep recovery after 12h (p=0.024), with a significant difference between the control and the environment (p=0.011). (3) compared with the normal control group and control group, C57BL6 mice after subacute partial sleep deprivation for 2 weeks in both cerebral cortex and hippocampus decreased sAPP levels were statistically significance (P0.05), but no statistically significant difference between the concentrations of cortex and hippocampus sAPP alpha (P0.05). (4) sleep deprivation can influence S D rats in Morris water maze spatial memory (the percentage of swimming time in the target quadrant) capacity (p=0.000), intraventricular injection of recombinant alpha sAPP can improve the spatial memory impairment (p=0.037), this improvement is more significant in 2 after injection (p=0.005). (5) compared with healthy controls, reduce AD patients and patients with chronic insomnia were sleep efficiency (p=0.000), increased awakening during sleep (p=0.000) and REM (p=0.000); sleep decreased in patients with AD increased the awakening time of patients with chronic insomnia (p=0.018), and patients with chronic insomnia sleep latency was significantly prolonged (p=0.000). (6) AD in cerebrospinal fluid of patients with sAPP levels than patients and healthy controls (decrease of chronic insomnia p=0.000, p=0.000), no significant difference between (P0.05). (7) sAPP alpha level and cerebrospinal fluid in patients with AD cognitive scale score and sleep efficiency has a significant positive correlation (Spearman 's rho0.676, p=0.016; 0.935, p=0.000 (8). ) dexzopiclone intervention in the treatment of insomnia can significantly improve the sleep parameters of PSG (P0.05). (9) dexzopiclone intervention can improve the cognition of patients with Chronic Insomnia Scale score (p=0.047), but does not change the CSF levels of sAPP (p=0.142); intervention therapy can increase AD cerebrospinal fluid levels of sAPP alpha (p=0.004). But the cognition of the patients with AD score did not improve significantly (p=0.514).
[Conclusion] first, sAPP alpha showed circadian changes in normal sleep wake state, reached the peak in the sleep period. Both acute or subacute complete sleep deprivation of sleep deprivation can lead to decrease sAPP levels in the brain, acute sleep deprivation led to decreased can be rapidly recovered after re sleep after recovery, has the correlation suggested that sAPP alpha level sleep and the brain. Secondly, intracerebral injection of recombinant sAPP alpha can memory against acute total sleep deprivation in the animal model of damage. Third, the levels of sAPP alpha in cerebrospinal fluid of AD patients was positively correlated with cognitive decline, score and sleep time, there are also symptoms of insomnia insomnia patients with chronic sAPP alpha level normal. Drug intervention to improve the symptoms of insomnia, AD in cerebrospinal fluid of patients with sAPP levels increased, but cognitive score no significant recovery, but for insomnia intervention can improve the treatment of chronic insomnia patients The score of cognition indicates that there is a correlation between the level of sAPP alpha in the human brain and sleep. The improvement of sleep can increase the level of sAPP alpha in the brain. But for those who have developed Alzheimer's disease, the improvement of sleep can not reverse the cognitive impairment.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

【引证文献】