Tau蛋白过度磷酸化相关的七氟醚麻醉后老年小鼠认知功能障碍的机制研究

发布时间：2018-04-29 20:33

本文选题：七氟醚 + 术后认知功能障碍　；参考：《复旦大学》2014年硕士论文

【摘要】：目的老年患者在手术麻醉后易罹患术后认知功能障碍(postoperative cognitive dysfunction, POCD),但其发病机制仍不清楚。最近有动物研究显示海马tau蛋白的过度磷酸化可能在全麻药物诱发的认知功能损伤中扮演重要的角色,但其具体机制尚不明确。本研究的目的是探讨成熟神经元细胞重新进入细胞周期是否参与七氟醚暴露后海马神经元内tau蛋白过度磷酸化诱发认知功能损伤的过程。方法第一部分：30只12月龄雄性C57BL/6J小鼠随机分为两组：七氟醚组(SEV组,n=15),吸入2.5%七氟醚+100%氧气2h；对照组(CON组,n=15),吸入100%氧气2h。七氟醚吸入结束后第七天,每组选取10只小鼠进行水迷宫实验,最终每组各有8只小鼠完成水迷宫试验；同时每组选取5只小鼠,取海马行western blot,测定其海马中总tau蛋白、磷酸化tau蛋白、cyclin D1、p27kipl及c-caspase-3的表达水平。第二部分：取40只出生24h之内的C57BL/6J小鼠,用于海马细胞培养。培养满15天后,将其分为四组：对照组(CON组),暴露于含95%02+5%C02的新鲜气体2h；七氟醚组(SEV组),在输入培养箱的以上新鲜气流中加入2%的七氟醚,暴露2h;LiCl组(Li组),在暴露于含95%02+5%CO2的新鲜气体前1h加入LiCl (20mmol/L)进行预处理；LiCl+-七氟醚组(Li+SEV组),在暴露于含2%七氟醚的新鲜气体前1 h加入LiCI(20mmol/L)进行预处理。气体暴露结束后收取细胞样本,采用western blot法测定各组磷酸化tau蛋白及cyclin D1的表达量。结果第一部分：七氟醚暴露可导致老年小鼠出现空间学习记忆能力损伤,同时增加了其海马中总tau蛋白、磷酸化tau蛋白、裂解的caspase-3和cyclin D1的表达,但对p27kip1的表达水平没有影响。第二部分：与CON组相比,SEV组磷酸化tau蛋白和cyclin D1表达水平明显升高,提示2h的七氟醚暴露可促进小鼠海马细胞tau蛋白的过度磷酸化,并增加cyclin D1的表达；与SEV组相比,Li+SEV组的磷酸化tau蛋白明显下降的同时,cyclin D1的表达亦下降,提示当tau蛋白的磷酸化被GSK-3β抑制剂阻断后,cyclinDl的表达随之下降,cyclinDl可能是磷酸化tau蛋白作用的靶点。结论七氟醚可引起老龄小鼠认知功能的损伤,这可能与其引起海马tau蛋白过度磷酸化有关；海马成熟神经元细胞重新进入细胞周期而不能完成细胞周期并最终走向凋亡可能是其重要的中间环节。
[Abstract]:Objective postoperative cognitive dysfunction (POCDD) is easy to occur in elderly patients after anesthesia, but the pathogenesis is still unclear. Recent animal studies have shown that excessive phosphorylation of hippocampal tau protein may play an important role in cognitive impairment induced by general anesthesia, but its mechanism is unclear. The aim of this study was to investigate whether the reentry of mature neuron cells into cell cycle is involved in the process of cognitive impairment induced by excessive phosphorylation of tau protein in hippocampal neurons after sevoflurane exposure. Methods in the first part, 30 12 month old male C57BL/6J mice were randomly divided into two groups: sevoflurane group (n = 30) and control group (n = 30), which inhaled 2.5% sevoflurane 100% oxygen for 2 h and 100% oxygen for 2 h in control group. On the seventh day after sevoflurane inhalation, 10 mice in each group were selected for water maze test, 8 mice in each group completed the water maze test, and 5 mice in each group were selected for western blot to determine the total tau protein in the hippocampus. Expression of phosphorylated tau protein cyclin D1 p27kipl and c-caspase-3. Part two: 40 C57BL/6J mice within 24 hours were selected for hippocampal cell culture. After 15 days of culture, they were divided into four groups: control group (Con) exposed to fresh gas containing 95 ~ 52 for 2 h, sevoflurane group (SEV) added 2% sevoflurane into the fresh air stream above the incubator. After 2 hours of exposure to fresh gas containing 95 5%CO2, 20 mmol / L LiCl was added to the liCl group, and 20 mmol / L LiCl-sevoflurane group was added to the fresh gas containing 2% sevoflurane one hour before exposure to the fresh gas containing 2% sevoflurane. The expression of phosphorylated tau protein and cyclin D1 were measured by western blot assay. Results in the first part, sevoflurane exposure induced spatial learning and memory impairment in aged mice, and increased the expression of total tau protein, phosphorylated tau protein, caspase-3 and cyclin D1 in hippocampus. But there was no effect on the expression of p27kip1. The second part: compared with the CON group, the phosphorylated tau protein and cyclin D1 expression level in the sevoflurane group were significantly increased, suggesting that sevoflurane exposure for 2 hours could promote the excessive phosphorylation of tau protein and increase the expression of cyclin D1 in the hippocampal cells of mice. Compared with SEV group, the phosphorylated tau protein of Li SEV group decreased significantly and the expression of cyclin D1 decreased, suggesting that the expression of cyclin D1 decreased after the phosphorylation of tau protein was blocked by GSK-3 尾 inhibitor. Cyclin D1 may be the target of phosphorylated tau protein. Conclusion sevoflurane can induce cognitive impairment in aged mice, which may be related to excessive phosphorylation of tau protein in hippocampus. Hippocampal mature neuron cells re-enter the cell cycle and fail to complete the cell cycle and eventually to apoptosis may be an important intermediate link.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614

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