严重烫伤合并铜绿假单胞菌脓毒症家兔外周血淋巴细胞蛋白质组学的研究

Proteomic study of peripheral blood lymphocytes of rabbits with severe burn and Pseudomonas aeruginosa sepsis

目的研究烫伤脓毒症时外周血淋巴细胞蛋白质组的变化特点。方法24只雄性成年家兔被随机分为对照组、烫伤组、烫伤脓毒症2h和6h组，每组6只。采用30％总体表面积（TBSA）I度烫伤模型，烫伤后24h经耳缘静脉注射铜绿假单胞菌悬液（ATCC27853，浓度6×10^12cfu／L）1ml／kg制备烫伤脓毒症模型；对照组用37℃温水进行假烫伤。烫伤组于伤后24h、烫伤脓毒症组于注射菌液后2h和6h取颈动脉血分离淋巴细胞，裂解细胞，提取总蛋白，用双向凝胶电泳分离蛋白质，考马斯亮蓝染色，扫描、分析图像并筛选差异蛋白质点（判断差异的标准为平均表达量相差超过3倍，由本课题组设定）。应用基质辅助激光解吸／电离飞行时间质谱分析方法得到肽质量指纹图谱，利用Mascot软件搜索数据库鉴定蛋白质。结果对照组、烫伤组、烫伤脓毒症2h和6h组平均蛋白质点数分别为1051±21、1026±30、1078±36、1065±31，平均匹配率分别为91％、89％、92％、94％。烫伤脓毒症2h与6h组间各蛋白质点表达量无差异；与对照组比较，烫伤组、烫伤脓毒症2h和6h组共筛选出19个差异蛋白质点，得到鉴定的有12个，包括11种蛋白质，即Cofilin、肽酰-脯氨酰-顺反式异构酶胞溶质蛋白A、泛素、核苷二磷酸激酶、谷氨酸脱氢酶、硒结合蛋白I、β-肌动蛋白、过氧化物氧化还原素6、膜联蛋白I、肌动蛋白-3、细胞视黄酸结合蛋白。结论严重烫伤并脓毒症时外周血淋巴细胞蛋白质组发生变化，表达改变并经鉴定的11种蛋白质功能涉及蛋白质折叠、装配、运输、降解，以及胞内信号传递、炎症、免疫、能量代谢、细胞增殖、分化、凋亡等，这些蛋白质与烧伤后脓毒症的发生有关。

Objective To study the effect of severe burn and Pseudomonas aeruginosa sepsis on the proteomics of lymphocytes （LCs） of rabbits. Methods Twenty-four rabbits were divided into four groups, i.e. control, severe scald, severe scald and 2-hour sepsis, severe scald and 6-hour sepsis （6 rabbits in each group）. The scald in rabbits was third degree in depth involving 30% of total body surface area （TBSA）. The sepsis model was reproduced by intravenous injection of a suspension of Pseudomonas aeruginosa （ATCC 27853, 6 × 10^12 efu/L） 1 ml/kg 24 hours after scald. The rabbits in control group were treated with warm water of 37 ℃. Peripheral blood was obtained from the carotid artery 24 hours after scald, or 2 hours after sepsis, or 6 hours after sepsis. The LCs in each blood sample were separated, disrupted and the total proteins of LCs were extracted. The proteins were separated by two dimensional gel electrophoresis. The gels were stained with Coomassie brilliant blue and then were scanned. The images were analyzed by PD quest software. The protein spots of discrepant expression were sieved and then analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS）. The peptide mass fingerprintings （PMFs） were obtained and were inputted into the data bank of proteins for identification of the proteins. Results The average spots of 6 gels were 1 051±21 （control）, 1 026±30 （severe scald）, 1078± 36 （2-hour sepsis） and 1 065± 31 （6-hour sepsis）, and the average matching rate were 91% （control）, 89% （severe scald）, 92% （2-hour sepsis） and 94% （6-hour sepsis）, respectively. No difference was found in the protein expression of LCs between 2-hour sepsis group and 6-hour sepsis group, but the protein expression of LCs in severe scald group, 2-hour sepsis group and 6-hour sepsis group differed when compared with control group. Nineteen protein spots expressed discrepantly were sieved and their PMFs were obtained. Twelve protein spots （including 11 proteins） were identified, including Cofilin, peptidylprolyl cis-trans isomerase cyclophilin A, ubiquitin, nucleoside diphosphate kinase, glutamate dehydrogenase, selenium binding protein I , β-actin, peroxiredoxin-6, annexin I , actin-3, cellular retinoic-acid binding protein. Conclusion The proteomics of peripheral blood LCs alters in rabbits with severe burn and Pseudomonas aeruginosa sepsis. The proteins with discrepant expression included 11 proteins, which are related with the folding, assembling, transportation and degradation of proteins, signal transmission, inflammation, immunization, energy metabolism, the proliferation, differentiation and apoptosis of cells. These proteins might be associated with the pathogenesis of severe burn and Pseudomonas aeruginosa sepsis.