【作者】 欧阳文；

【导师】 刘浔阳；

【作者基本信息】 中南大学 ， 外科学， 2006， 博士

【摘要】 目的应用“Y”迷宫训练检测异氟醚吸入全身麻醉后老龄大鼠学习能力和记忆功能变化，采用双向凝胶蛋白电泳结合质谱分析技术筛选和鉴定相应情况下海马蛋白质组表达差异，旨在进一步确定异氟醚麻醉后老龄大鼠记忆和学习功能的变化情况，探讨异氟醚对老龄大鼠海马蛋白质组是否有影响以及影响程度，尽可能找出与麻醉后认知功能改变相关的特异蛋白。方法（1）．本课题的第1阶段：①．随机将75只22月龄SD大鼠分为C组（对照组，n=35）和Ⅰ组（异氟醚组，n=40）。C组吸入含40％氧气的空氧混合气体2小时。Ⅰ组以3％异氟醚＋40％氧气吸入诱导，随后以1.2％异氟醚＋40％氧气维持全身麻醉2小时。②．两组大鼠于吸氧对照或麻醉苏醒后24小时开始应用“Y”型迷宫连续3天训练检测其学习记忆功能，每天训练20次，记录正确反应次数、全天总反应时间、主动回避次数和三天学会率。③．麻醉结束后24小时和72小时分别于两组中随机抽取5只大鼠，取左侧海马-80℃冻存。取右侧海马行尼氏染色和电镜观察。（2）．本课题的第2阶段：取冻存的左侧海马组织进行匀浆、裂解、水化制备蛋白溶液样品，应用双向凝胶电泳获取含有蛋白质斑点的凝胶，然后考染显色，扫描仪扫描获取图像，运用软件识别、合成、匹配，分析组间差异，切取凝胶中差异蛋白点，胰酶消化分解，ZipTip^TM脱盐，经质谱分析，获取肽指纹图，登陆互联网查询结果以鉴定差异蛋白点。结果①．麻醉后24小时Ⅰ组正确反应次数为10.97±3.48次，小于C组13.57±2.62次（P＜0.01）；主动回避次数为1.11±1.07次，小于C组3.20±2.59次（P＜0.01）。麻醉后48小时Ⅰ组正确反应次数为13.53±3.42次，小于C组16.07±2.79次（P＜0.01）；主动回避次数为1.27±1.56次，小于C组6.45±4.97次（P＜0.01）；全天总反应时间Ⅰ组81.93±45.01秒，大于C组59.27±26.37秒（P＜0.05）。麻醉后72小时两组间各项观察指标无差异显著性。三天学会率C组42.86％，Ⅰ组31.58％，Ⅰ组低于C组（P＜0.05）。②．两组大鼠尼氏染色后未发现海马组织神经元有变性坏死现象；高倍电镜下可见两组大鼠海马神经元基本结构完整，与C组相比较，麻醉结束后24小时Ⅰ组突触后膜致密物质有变薄趋势，突触间隙增宽，这些变化在麻醉结束后72小时恢复正常。③．获得了较满意的双向凝胶电泳胶图，图象分析结果显示C_24h组凝胶蛋白质点为906±46，I_24h组凝胶为913±51，C_72h组凝胶蛋白质点为936±36，I_72h组凝胶蛋白质点为928±44。C_24h和I_24h组差异蛋白质点共有17（5／12）个；C_72h和I_72h组差异蛋白质点共有16（12／4）个，MALDI-TOF-MS分析以后鉴定出22种蛋白质。结论（1）．老龄大鼠吸入1.2％异氟醚2小时后早期学习记忆能力下降，这一现象可能和海马CA3区突触界面结构改变相关，但是麻醉结束后72小时这些变化基本正常。（2）．老龄大鼠吸入1.2％异氟醚2小时后24小时海马蛋白质组发生了变化，其中神经元信号传导和突触前膜囊泡释放相关的蛋白质可能参与了老龄大鼠异氟醚麻醉后学习记忆功能变化，不过这些蛋白质的表达差异在麻醉后72小时内都逐渐消失。更多还原

【Abstract】 Objective To investigate the changes of leaning and memory function after isoflurane anesthesia with Y-maze training, then to measure the alteration of proteome of brain hippocampus after isoflurane anesthesia in aged rats by the means of two dimensional gel electrophoresis and MALDI-TOF-MS in order to demonstrate the effects of isoflurane on cognition and seek the proteins related to the alteration of leaning and memory function after isoflurane anesthesia. We wish to find some kinds of special proteins related with cognitive function as far as possible.Methods The first stage of the experiment①)To randomly divided 75 22-month-old SD rats into group contrast （group C, n=35） and group isoflurane （group I, n=40） . The rats in Group C only inhaled mixed gas containing 40% oxygen for two hours. The rats in group I were induced by inhalation of 3% isoflurane combined 40% oxygen. Then the general anesthesia was maintained with 1.2% isoflurane combined 40% oxygen for two hours.②. We use Y-maze training to test the learning and memory function of aged SD rats from 24 hours after waking up（ Group I） or inhalation of 40% oxygen（group C）. The training time is 3 days and 20. times per day. The number of correct reaction, total reactive time, the number of initiative avoidance and the rate of acquire the task in 3 days are recorded.③. Five rats randomly selected from each group at 24h after stopping oxygen (group C_24h) or isoflurane (group I_24h) inhalation. And five rats randomly selected from each group at 72h after stopping oxygen (group C_72h) or isoflurane (group I_72h) inhalation. The left hippocampus of the aged rats mentioned above was cut down and reserved in - 80℃. The right hippocampus of the aged rats was cut down for Nissl’s staining and observation of electron microscope.The second stage of the experiment: The left hippocampus was made into protein liquor by the means of homogenate, schizolysis and hydration. By the means of two dimensional gel electrophoresis, we got some pieces of gel containing protein spots. The Commassie’s blue staining were used to make the gel coloration. We analyzed the images with scanning, discerning, synthesizing and matching, and we got the different protein spots among the groups. The different protein spots in the gel were removed and analyzed by the means of trypsinization, ZipTip^TM desalinization and mass chromatographic analysis. So we got peptides mass fingerprints, which were analyzed by using database and software from internet to identify proteins.Results①The number of correct reaction in I₂₄ is 10.97±3.48, which is less than 13.57±2.62 in C₂₄ （P＜0.01）; the number of initiative avoidance in I₂₄ is 1.11±1.07, which is less than 3.20±2.59 in C₂₄ （P＜0.01）. The number of correct reaction in I₄₈ is 13.53+-3.42, which is less than 16.07±2.79 in C₄₈（P＜0.01）; the number of initiative avoidance in I₄₈ is 1.27±1.56, which is less than 6.45±4.97 in C₄₈ （P＜0.01）; the total reactive time in I₄₈ is 81.93±45.01 sec, which is more than 59.27±26.37 sec in C₄₈ （P＜0.05）. There is no significant difference between I₇₂ C₇₂ in every index. The rate of acquire the task in 3 days in group C is 42.86%, which is more than 31.58% in groupⅠ（P＜0.05）.②There were no apomorphosis and necrosis in hippocampus with Nissl’s staining. With electron microscope we found the postsynaptic density trend to thin and synaptic cleft trend to widen after isoflurane anesthesia, but there was no disorganization in the hippocampal neurone.③We got some pieces of ideal gel per group. The results showed that there were 906±46 protein spots in group C_24h, 913±51 protein spots in group I_24h, 936±36 protein spots in group C_72h, 928±44 protein spots in group I_72h, The results also showed that there were 17 （5/12） different protein spots between group I_24h and group C_24h, and there were 16 （12/4） different protein spots between group I_72h and group C_72h. Twenty two protein spots of all spots were identified by the means of MALDI-TOF-MS.Conclusions1. The results demonstrate that general anesthesia with 1.2% isoflurane for 2 hours may result in cognitive impairment in the aged rats, and this phenomenon may related to change of synaptic interface in CA3 area of hippocampus , but all of the changes almost recovery 72 hours after the anesthesia.2. The study shows that general anesthesia with 1.2% isoflurane for 2 hours may result in the alteration of proteome of hippocampus in aged rats. The changes of signal conduction and synapse correlated proteins may related to cognitive impairment in the aged rats, but the change of expression of the proteins may recovery 72h after the anesthesia.更多还原