严重烫伤合并铜绿假单胞菌脓毒症影响家兔外周血中性粒细胞的蛋白质组学研究  

Proteomic study of polymorphonuclear leukocytes in peripheral blood of rabbits with severe burns and fei, HUANG Xiao-yuan. Changsha 410008, China sepsis

作　　者：曾纪章[1] 张丕红[1] 李莉莉[1] 任利成[1] 梁鹏飞[1] 黄晓元[1] 

机构地区：[1]中南大学湘雅医院烧伤整形外科,长沙410008

出　　处：《中华急诊医学杂志》2008年第2期123-127,共5页Chinese Journal of Emergency Medicine

基　　金：国家自然科学基金资助项目（30571920）

摘　　要：目的研究严重烫伤合并铜绿假单胞菌脓毒症家兔外周血中性粒细胞蛋白质组变化。方法实验地点在卫生部肿瘤蛋白质组学重点实验室（湘雅医院内），实验动物为健康雄性成年中国本兔24只，随机分为四组（每组6只）：对照组（A组）、严重烫伤组（B组）、严重烫伤并脓毒症2h组（C组）和严重烫伤并脓毒症6h组（D组）。A组37℃温水假烫，B组烫伤（30％TBSA，Ⅲ度）后24h采颈动脉血分离中性粒细胞，C、D组烫伤（30％TBSA，Ⅲ度）后24h经耳缘静脉注射对数生长期铜绿假单胞菌悬液（ATCC27853，浓度6×10^9cfu／ml，剂量1ml／奴），再分别于2、6h后采颈动脉血分离中性粒细胞。裂解细胞，提取总蛋白。利用双向凝胶电泳分离蛋白质，考马斯亮蓝染色，扫描图像，PDQuest软件分析图像并筛选组间差异蛋白质点（判断差异的标准为平均表达量相差超过3倍，由本课题组成员设定）。再从凝胶上切割差异蛋白质点，胰酶消化，应用基质辅助激光解吸／电离飞行时间质谱分析，得到肽质量指纹图，利用Mascot软件搜索数据库、鉴定蛋白质。结果A、B、C、D组平均蛋白质点数分别为（759±20）、（749±27）、（786±29）、（774±26）个，平均匹配率分别为93％、91％、89％、92％。C、D两组间蛋白质点表达量差异无统计学意义，B、C、D组与A组比较，筛选出21个差异蛋白质点，鉴定了其中9个，包括7种蛋白质。结论家兔严重烫伤并铜绿假单胞菌脓毒症时外周血中性粒细胞蛋白质组发生变化，表达改变的有蛋白质二硫键异构酶、β-肌动蛋白、膜联蛋白Ⅰ、巯基特异性抗氧化蛋白等，功能涉及细胞内酶活性、细胞能动功能及变形性、炎症、代谢、抗氧化能力、细胞凋亡等，这些蛋白质与烧伤后铜绿假单胞菌脓毒症有关。Objective To study the proteome change of polymorphonuclear leukocytes （PMNs） of rabbits with severe burn and Pseudomonas-aeruginosa sepsis. Method Twenty-four rabbits were divided into four groups including A （control）, B （severe scald）, C （severe scald and sepsis 2 h） and D （severe scald and sepsis 6 h）. The rabbits in group A （fake scald） and group B （30% TBSA, Ⅲ° burn） were operated 24 h later and the peripheral blood was drawn from carotid artery. The rabbits in group C and group D were transfused with the suspension of Pseudomonas-aeruginosa （ATCC 27853） 24 h after scald （30%TBSA, Ⅲ° burn） and the peripheral blood samples were obtained 2 h （ C ） or 6 h （D） later from the carotid artery. The PMNs were separated from the blood. The total proteins from the destroyed cells were extracted. Proteins were separated by using 2- dimensional electrophoresis （2-DE）. Gels were stained with eoomassie brilliant blue and scanned. The images were analyzed with PDquest software. The protein spots expressed discrepantly were sieved. The peptide mass fingerprints of them were obtained by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and then imputed into the data bank of proteins for identification with Mascot soft-ware. Results The clear, high-resolution 2-DE images of PMNs of rabbits were obtained. The spots in average of each group were 759 ± 20 （A）, 749 ± 27 （B）, 786 ± 29 （C） and 774 ± 26 （D）. The matching rate in average of each group were 93% （A）, 91% （B）, 89% （C） and 92% （D）. There was no obvious difference found in the expression of PMNs protein between C and D. The expression of PMNs protein in groups of B, C and D differed from that in group A. Twenty-one spots expressed discrepantly were sieved and their peptide mass fingerprints were obtained. Nine protein spots from 7 proteins were identified. Condusions The proteome of PMNs altered in rabbits with severe burns and Pseudomonas-aeruginosa

关 键 词：兔 烧伤 脓毒症 铜绿假单胞菌 中性粒细胞 蛋白质组 

分 类 号：R644[医药卫生—外科学] R459.7[医药卫生—临床医学]