【作者】 田首元；

【导师】 叶铁虎；

【作者基本信息】 中国协和医科大学 ， 麻醉学， 2010， 博士

【摘要】 研究背景术后认知功能障碍(postoperative cognitive dysfunction, POCD)是麻醉后中枢神经系统常见的并发症之一。导致POCD的因素很多,一般认为老龄是唯一明确的危险因素。研究显示,麻醉药可能是患者POCD的主要诱发因素之一。丙泊酚是临床常用的静脉麻醉药,具有诱导迅速、苏醒快,遗忘等优点,特别适用于各种门诊诊断治疗性操作的麻醉(比如,无痛胃镜)。氯胺酮是N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)受体的非特异性拮抗剂,在术中术后镇痛应用广泛。研究表明,麻醉剂量丙泊酚或氯胺酮均可能导致学习记忆或行为学的改变,但其潜在的机制尚不明确。海马是中枢神经系统内与学习记忆等认知功能最为密切相关的重要部位。很多导致海马损伤的因素均可能会造成认知功能障碍。蛋白质是生命活动的最主要和最直接的体现者和执行者,也是多种致病因素和药物作用的靶分子,因此深入研究蛋白质的组成和活动规律对解释生理和病理现象有非常意义。蛋白质组学研究是从生物大分子整体水平研究蛋白质表达谱和蛋白质与蛋白质之间相互作用的新兴领域。具有全面性、整体性、高通量、大规模的特点。近年来,蛋白质组学已经逐渐被用于麻醉学领域的研究,并且获得了很好的结果。但该方法用于丙泊酚或氯胺酮麻醉对认知功能领域的研究尚未见到。研究目的1.通过行为学实验,观察丙泊酚或氯胺酮麻醉对不同时间点(麻醉后第1天和第7天)老龄大鼠认知功能的影响；2.结合蛋白质组学研究方法和生物信息学方法分析老龄大鼠海马组织蛋白的差异表达情况(麻醉后第1天和第7天),初步分析丙泊酚或氯胺酮麻醉后海马蛋白网络的变化,尽可能找出其与认知功能影响相关联的蛋白,从整体上全面深入探讨丙泊酚或氯胺酮麻醉影响老龄大鼠认知功能的分子机制；3.检测老龄大鼠血清S100β蛋白、神经烯醇化酶(NSE)等生物标记物,初步评估其在诊断丙泊酚或氯胺酮麻醉是否影响认知功能中的预测价值。研究方法选用健康雄性Wistar大鼠,20月龄,体重560-610g,随机分为对照组(C组)和丙泊酚组(P组)或氯胺酮组(K组)。P组大鼠经腹腔注射丙泊酚60mg／kg(6ml／kg)实施麻醉,然后酌情追加,维持麻醉3 h。K组大鼠经腹腔注射丙泊酚80mg/kg(8ml／kg)实施麻醉,然后酌情追加,维持麻醉3 h。C组腹腔注射等量生理盐水。于停药后1天开始行跳台实验(第1和7天)和Morris水迷宫实验(连续7天)测试认知功能。分别于第1天和第7天实验结束后大鼠断头取脑分离海马组织,2DQuant蛋白定量试剂盒测定蛋白浓度后,取150μg蛋白／胶上样进行双向凝胶(2-DE)电泳分离蛋白样品。2-DE凝胶银染显色蛋白点,图像扫描并输入凝胶图像分析软件ImageMaster 2-D Platinum(V5.0)进行凝胶匹配找出两组不同时间点的差异表达蛋白。确认的差异表达蛋白点切胶消化,提交基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)进行分析,获得差异表达蛋白的肽质量指纹图谱(PMF)。利用相关数据库(NCBInr, Mascot)对PMF进行检索,初步确定差异蛋白身份。根据确认蛋白的生物信息,结合对应差异表达蛋白点在蛋白凝胶中的位置,确认质谱结果的正确性。另外,通过串联质谱(MALDI-TOF/TOF MS)对上述结果进一步验证。进一步利用生物信息学分析确定差异蛋白的亚细胞定位、参与的生物学过程和主要功能,初步分析丙泊酚或氯胺酮麻醉后海马蛋白网络的变化。另外,随机选择部分蛋白通过Western-blot观察其不同时间点的动态变化,用以验证2-DE凝胶电泳结果的可靠性。分别于麻醉后1天和7天抽血采用双抗体夹心酶联免疫吸附法(ELISA)检测S100p和NSE浓度。研究结果丙泊酚部分1.行为学实验：1)跳台实验：与C组比较,丙泊酚麻醉结束后1天时老龄大鼠学习记忆功能减退；学习阶段潜伏期、受电击总时间均明显延长,学习错误次数较多(P<0.01)；记忆阶段潜伏期缩短,而受电击总时间明显延长,记忆错误次数较多(P<0.01)。麻醉结束后7天时,两组间各指标差异没有统计学意义。2) Morris水迷宫实验：水迷宫定位航行实验结果表明,丙泊酚麻醉结束后第1天,P组大鼠逃避潜伏期和总游泳距离明显长于C组(P<0.01),而在第2-6天,与C组比较差异无统计学意义。与第1天比较,C组和P组其余各个时间点逃避潜伏期和总游泳距离均明显缩短(P<0.01)。两组大鼠平均游泳速度无统计学差异。水迷宫空间探索实验结果表明,两组大鼠原平台象限探索时间、探索距离、穿越平台次数间差异无统计学意义。2.2-DE凝胶电泳及蛋白质组学分析：与C组相比,丙泊酚麻醉后第1和7天分别发现21和15个差异表达蛋白点,其中分别有17和10个经MALDI-TOF成功鉴定；生物信息学分析表明,大部分检定蛋白分别主要位于细胞器(2天分别各有11个、5个),细胞浆(12、3),细胞核(3、3),细胞骨架(7、0),质膜(8、1)。检定蛋白分别主要具有催化功能(12、5),蛋白质折叠(2、0),生物结合(9、4),结构结构分子(5、1),分子伴侣(1、0)和抗氧化功能(1、0)。此外大部分蛋白分别主要参与代谢(6、6),生物调节(10、2),发生发育(10、1)以及刺激-反应过程(1、0)。通过Western-blot观察选定蛋白的差异表达,其变化趋势与双向凝胶电泳结果一致。3.两组大鼠药物处理后相应时间点血清S100β和NSE浓度差别没有统计学性意义。氯胺酮部分1.行为学实验和血清生化指标：结果变化趋势与丙泊酚部分相似。2.2-DE凝胶电泳及蛋白质组学分析：与C组相比,氯胺酮麻醉后第1和7天分别发现26和15个差异表达蛋白点,其中分别有21和8个经MALDI-TOF成功鉴定；生物信息学分析表明,大部分检定蛋白分别主要位于细胞器(2天分别各有14个、6个；下同),细胞浆(13、2),细胞核(5、2),细胞骨架(5、1),质膜(8、2)。检定蛋白分别主要具有催化功能(16、6),蛋白质折叠(1、0),生物结合(13、4),结构分子(4、1),转运(1、1),分子伴侣(1、0),第7天有2个蛋白分别参与抗氧化和转录调节。此外大部分蛋白分别主要参与代谢(11、5),生物调节(10、4),发生发育(15、4)以及刺激-反应过程(6、0)。通过Western-blot观察选定蛋白的差异表达,其变化趋势与双向凝胶电泳结果一致。结论丙泊酚或氯胺酮麻醉后可以导致第1天老龄大鼠认知功能障碍,但不影响第7天的认知功能。其机制可能与麻醉后第1天海马组织较多认知相关蛋白出现差异表达有关；差异表达的蛋白质呈现多维网络化联系,主要涉及多个生物学过程和多种功能,包括糖酵解和能量代谢,细胞骨架蛋白质量控制,突触囊泡转运和再循环、递质释放,抗氧化,Ca2+稳态,细胞内信号转导以及抗凋亡活性等,进而影响突触可塑性和学习记忆。NSE和S100β在预测丙泊酚或氯胺酮麻醉与认知功能关系上没有太大价值。更多还原

【Abstract】 BackgroundPostoperative cognitive dysfunction (POCD) is one of the common complications of central nervous system after anesthesia and surgery. In spite of many risk factors, increasing age (≥60) is the only definitely significant risk factor for the development of POCD. It is generally believed that, anesthetics may be one of the main factors inducing POCD. Propofol (2,6-diisopropylphenol) has the advantage of fast induction, better-quality recovery as well as amnesia and is widely used for induction and maintenance of general anesthesia as well as for procedural sedation(e.g. painless gastroscopy). Ketamine, which can produce analgesia, is as a noncompetitive antagonist to the phencyclidine site of the N-methylmethyl-D-aspartate (NMDA) receptor for the excitatory neurotransmitter glutamate. However, NMDA receptor-mediated long term potential (LTP) is believed to induce memory. Increasing evidence indicate that propofol or ketamine can produced amnesia and behavior disorder at anesthetic doses, but the precise molecular mechanisms underlying the cognitive impairment are poorly understood so far. Cognitive functions are known to involve multiple brain areas, complex processes and variable cellular components. It is demonstrated that hippocampus is one of the most important brain areas that participating in cognitive functions such as memory and learning. Many factors impairing hippocampus can cause cognitive dysfunction.Proteins are the most important biomacromolecules that directly embody and execute the function of life activities. They are also the target molecules that many causative factors and drugs bind and produce effects. Therefore, it is most significant to deeply explore the protein composition and activities pattern so as to elucidate the physiological and pathological process. The new method of proteomics analysis used to investigate changes in abundance of proteins has the advantage that high-capacity screening of proteins and integrally studying the differentially expressed protein profile and the interactions between proteins. Recently, proteomic tools have been used to explore the anesthetic effects, mostly focused on the inhalation anesthetics, and the results were enlightening. However, there is few study devoted to assess the effect of propofol on the cognitive function by proteomics analysis. Objective1. To observe the effects of propofol/or ketamine on cognitive function in aged rats at the 1st day and 7th day after termination of propofol/or ketamine anesthesia through behavioral experiments;2. To study the differentially expressed protein profile in aged rat hippocampus by proteomics analysis and bioinformatics tools, preliminarily understand the possible alternation of hippocampus proteins’network in post-propofol/or post-ketamine anesthesia was preliminary, try to explore the hippocampus protein mostly related to cognitive function and thus integrally deeply investigate the molecular mechanism of cognitive impairment after propofol/or ketamine anesthesia.3. To roughly investigate the value of S-100βand neuron specific enolase (NSE) in reflecting POCD after propofol/or ketamine anesthesia.MethodsMale Wistar rats aged 20 months weighing 560-610g were randomly divided into 2 groups:control group(C) and propofol group(P)/ketamine group(K). The rats in Group P or Group K received intraperitoneal(IP) propofol 60mg/kg(6ml/kg) or ketamine 80mg/kg(8ml/kg) firstly and were boosted half of the initial dose when righting reflex emerged, whereas the rats in Group C received equal volume normal saline instead of propofol/ketamine in same way. One day after termination of drug administration, the rats were assessed with Morris water maze Test 4 times a day for 7 consecutive days and Step-down Test (1st day and 7th day, respectively). The rats were killed by decapitation after the test was finished. The blood samples in each group were collected to test the concentrations of S100βand NSE by ELISA and the hippocampus was isolated immediately. Proteins extracted from rat hippocampus were quantified by the 2D Quant Protein Assay Kit and 150μg protein solution was loaded in an IPG gel strip for 2-dimensional electrophoresis (2-DE) analysis.2-DE gels were silver stained to colour the isolated protein spots. Gel images were scanned and input into the gel image analyzing software ImageMaster 2-D Platinum(V5.0) to match the gels of two groups, compare protein expression difference and screen for the differentially expressed protein spots. The differential expressed protein spots were cut off and digested by trypsin for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis to obtain corresponding peptide mass fingerprint (PMF) identities. Each protein were identified and confirmed by checking the biological information provided by MS and location of the corresponding protein spot in gel images. In addition, identification of MS results was also performed by MALDI-TOF/TOF MS. Bioinformatics analysis through Gene Ontology (GO) software was further used to summarize the subcellular distribution, molecular function and biological processes of the differentially-expressed identified proteins. The expression changes of selected protein were also assayed using Western-blot to verify the reliability of 2-DE results.ResultsPropofol fraction1. Behavioral experiments:(1) In the step-down test, aged rats in Group P showed significantly learning and memory impairment at the 1st day after propofol anesthesia compared with those in Group C. For post-propofol anesthesia aged rats, the latency, total time of electric shock and error-number in the learning phase increased significantly (p<0.01). The total time of electric shock and error-number displayed similar trend in the memory phase, while the latency decreased significantly (p<0.01). There were no differences between two groups at the 7th day after propofol anesthesia with respect to each index as above.(2) In Morris water maze, the escape latency and total swimming distance to find the submerged platform in Group P at the 1st day after propofol anesthesia were significantly longer than that of Group C (p<0.05 or 0.01). In the probe test, the two groups were comparable with respect to both time and distance of aged rats spent in target quadrant as well as number of crossing over the position.2.2-DE and Proteomics ananlysis: Compared with Group C,21 or 15 differentially expressed proteins were detected respectively on the 1st day or 7th day after propofol anesthesia, among which 17 or 10 proteins were successfully identified with MALDI-TOF MS. Gene Ontology(GO) analysis revealed that the numbers of identified proteins which mainly distributed in cell component were:11 or 5 (organelle); 12 or 3 (cytosol); 3 or 3 (nucleus); 7 or 0 (cytoskeleton); 8 or 1 (membrane), respectively. Functionally, most finally identified proteins were involved in catalytic activities (12,5 respective); binding activities (9,4); protein folding(2,0); structural molecule(5,1); chaperone(1,0) and antioxidant(1,0). According to biological process category,6 proteins were involved in metabolic process respectively; 10 or 2 proteins were involved in biological regulation; 10 or 1 were involved in developmental process; 1 protein was involved in stimulus-response on the 1st day after propofol anesthesia while none was on the 7th day. Change patterns of 2 selected proteins in 2-DE were identical with those in Western blot.3. Two groups were comparable with respect to both the concentrations of S100βprotein and NSE.Ketamine fraction1. Behavioral experiments: The results of step-down test and Morris water maze test in ketamine fraction displayed a similar trend with propofol fraction.2.2-DE and Proteomics ananlysis: Compared with Group C,26 or 15 differentially expressed proteins were detected respectively on the 1st day or 7th day after propofol anesthesia, among which 21 or 8 proteins were successfully identified with MALDI-TOF MS. Gene Ontology(GO) analysis revealed that the numbers of identified proteins which mainly distributed in cell component were:14 or 6 (organelle); 13 or 2 (cytosol); 5 or 2 (nucleus); 8 or 2(membrane); 5 or 1 (cytoskeleton), respectively. Functionally, most finally identified proteins were involved in catalytic activities (16,6 respective); binding activities (13,4); protein folding(1,0); structural molecule(4,1); tansport(1,1) and chaperone(1,0). According to biological process category,11 or 5 proteins were involved in metabolic process; 10 or 4 proteins were involved in biological regulation; 15 or 4 proteins were involved in developmental process; 6 proteins were involved in stimulus-response on the 1st day after propofol anesthesia while none was on the 7th day. Change patterns of 2 selected proteins in 2-DE were identical with those in Western blot.3. Two groups were comparable with respect to both the concentrations of S100βprotein and NSE.ConclusionPropofol or ketamine can cause the cognitive dysfunction in aging rats on the 1st day after anesthesia whereas has no effect on that of 7th day. The mechanism may involved in the multiple differentially expressed proteins of hippacampus in the 1st day which induced by propofol or ketamine anesthesia. The differentially expressed proteins show multidimensional network relationship with each other. Multiple processes and variable functions in rat hippocampus are involved which impair the synapse plasticity and memory, including glycolysis and energy metabolism, protein synthesis and protein qulity control, cytoskeleton morphology and dynamics, synaptic vesicle transport and recycle, transmitter release, antioxidation, Ca2+ homeostasis, signal transduction and apoptosis. NSE and S100βprotein, serving as biochemical markers, appear to be of limited value in detecting POCD after propofol or ketamine anesthesia.更多还原