中性粒细胞体外黏附迁移滞留后蛋白质组变化.doc.doc

中性粒细胞体外黏附迁移滞留后蛋白质组变化 中南大学湘雅医院 张丕红 杨柳荣 吕春柳 梁鹏飞 曾纪章 任利成 黄晓元 【摘要】目的 观察中性粒细胞体外黏附迁移、滞留后蛋白质组的变化，探讨中性粒细胞在烧伤后炎症反应中的病理生理机制，寻找烧伤炎症的分子标志。方法 采集健康志愿者外周血(分离中性粒细胞和正常血清)、特重度烧伤患者血清和Ⅱo烧伤创面水泡液，运用Transwell建立中性粒细胞与人脐静脉血管内皮细胞体外黏附迁移模型，并将黏附迁移后的中性粒细胞在烧伤水泡液中培养建立滞留模型，收集黏附迁移后和滞留后中性粒细胞，以正常中性粒细胞做对照，进行二维凝胶电泳、应用PDQuest软件筛选差异表达蛋白点、辅助激光解析电离飞行时间质谱分析获取肽质量指纹图谱，Mascot查询软件搜索数据库，鉴定特征性差异表达蛋白。结果 与正常中性粒细胞比较，黏附迁移、滞留后中性粒细胞蛋白质组学分析，有33个显著差异表达的蛋白点,鉴定出22个蛋白点16种蛋白质。结论中性粒细胞体外黏附迁移及滞留后蛋白质组发生了明显变化，差异表达蛋白质可能在烧伤后炎症失控、免疫紊乱、细胞凋亡延迟、血管通透性增加中起一定作用。 【关键词】中性粒细胞，人脐静脉血管内皮细胞，烧伤血清，烧伤水泡液，蛋白质组学 基金项目：国家自然科学基金资助项目 【ABSTRACT】OBJECTIVE To observe the proteomics changes of polymorphorphonuclear neutrophils trans-endothelial migration and sequestration in burn blister fluid in vitro, PMN trans-endothelial migration and sequestration model were built and proteomics analysis was used to identify the biomarkers of inflammation after burn injury and explore the pathogenesis of burn sepsis. METHODS Normal human PMN and serum were harvested from healthy volunteers, burn serum isolated from massive burn patients within 48 hrs after burn injury, and burn blister fluid was collected from intact blisters on burn wound within 24 hrs. PMN transmigration followed adhesive via HUVEC stimulated with burn serum and sequestration in burn blister fluid model were established using transwellsTM inserts. The total proteins of PMN in healthy volunteers (A group), transmigration followed adhesive (B group), sequestration in burn blister fluid (C group) were separated by immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis，and these images were analyzed by PDQuest 7.1.0 software. Discrepant expression protein spots were analyzed by Matrix-assisted laser desorption/ionization time of flight mass spectrometry and peptide mass fingerprinting obtained and searched in database to identify of the proteins. RESULTS 33 differentially expressed proteins spot were found between normal PMN group and the others groups, and