丙泊酚影响大鼠海马难溶成分的比较蛋白质组学.docVIP

中青年优秀论文评奖 丙泊酚影响大鼠海马难溶成分的比较蛋白质组学研究 张雪娜 岳云 摘要 目的 观察丙泊酚麻醉后不同时间点，大鼠海马组织难溶成分蛋白表达的动态变化，初步分析蛋白网络的改变，深入探讨丙泊酚影响认知功能的分子机制。方法 健康雄性Wistar大鼠60只，随机分为对照组和实验组（丙泊酚麻醉3小时后1h、6h、24h、7d组，每组10只）；大鼠断头取脑分离海马组织，提取蛋白样品。采用双向凝胶（2-DE）电泳分离蛋白样品，生物质谱（MALDI-TOF）鉴定差异表达蛋白点。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。随机选择部分差异蛋白，通过Western-blot验证双向电泳结果。并采用RT-PCR分析部分差异蛋白在mRNA水平的变化。 结果 与对照组相比，丙泊酚麻醉后不同时间点内共发现59个差异表达蛋白，表现为多种动态变化趋势；其中43个经MALDI-TOF成功鉴定。蛋白表达变化主要集中于麻醉后24h内。生物信息学分析表明，大部分检定蛋白位于细胞器（34个）和膜（19个）；主要具有结合（41个）和催化功能（31个）；参与代谢（27），调节（21），发生发育（19）以及刺激-反应过程（14）。通过Western-blot观察SEPT5和SOD2的差异表达，其变化趋势与双向凝胶电泳结果一致。部分差异蛋白mRNA水平的变化与蛋白表达情况不一致。结论 丙泊酚麻醉后，部分海马组织难溶蛋白出现差异表达，蛋白质网络发生多维、动态改变；其动态特征与丙泊酚麻醉后认知功能损害的时程一致；但这些蛋白及相关生物学过程在认知功能改变中的具体作用有待深入探讨。 关键词：丙泊酚；难溶蛋白；蛋白质组；海马 作者单位：首都医科大学附属北京朝阳医院麻醉科 Proteomic Profiling of the Insoluble Fractions in the Rat Hippocampus Post Propofol Anesthesia Abstract Objective Propofol is known to produce amnesia but the underlying mechanisms are not clear. Current study was designed to observe dynamic changes in insoluble protein expressions post propofol anesthesia in rat hippocampus, trying to explore possible mechanisms of cognitive impairments. Methods Proteins extracted from rat hippocampus were separated by 2-dimensional electrophoresis (2-DE). Their expression patterns were observed at 1, 6, 24 hours and 7 days after 3 hours of propofol anesthesia. Differential expressed protein spots among groups were submitted to matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOF MS) assay and peptide mass fingerprinting (PMF) identification. All identified proteins were also analyzed through Gene Ontology (GO). The change patterns of expression of selected proteins were further assayed using Western blot and RT-PCR. Results Fifty-nine differentially expressed proteins were detected, among which 43 were successfully identified with MALDI-TOF MS. GO annotation revealed that identified proteins mainly distributed in membrane (19) and organelles (34). Functional