严重烫伤影响家兔外周血中性粒细胞的蛋白质组学研究

Proteome Study of Peripheral Blood Polymorphonuclear Leukocytes in Rabbits with Severe Burns

【目的】研究严重烫伤对家兔外周血中性粒细胞蛋白质组的影响。【方法]12只家兔分为A、B两组（每组6只），A组为对照组（假烫处理）、B组为严重烫伤组（30％TBSA，II度）。A组假烫、B组烫伤后24h经颈动脉采血分离中性粒细胞。裂解细胞，提取蛋白质。利用固相pH梯度胶条（240mm×3mm×0．5mm，pH3～10）及SDS-PAGE双向凝胶电泳分离蛋白质，考马斯亮蓝染色，图像扫描，PDQuest软件分析图像，筛选出差异表达蛋白质点。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，再利用Mascot软件搜索数据库，鉴定蛋白质。【结果】建立了家兔外用血中性粒细胞蛋白质2-DE图谱。A、B组平均蛋白质点数分别为（759±20）个、（749±27）个，平均匹配率分别为93％、9l％。两组间筛选出19个差异蛋白质点，鉴定了其中8个，包括7种蛋白质。【结论】家兔严重烫伤后外同血中性粒细胞蛋白质组发生改变，表达变化的蛋白质包括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、细胞能动功能及可变形性、炎症、代谢、抗氧化能力、细胞凋亡等，这些蛋白质与严重烧伤后中性粒细胞功能改变有关。

[Objective] To study the effect of severe burns on the protein expression of polymorphonuclear leukocytes（PMNs） in rabbits. [Methods] Twelve rabbits were divided into two groups including group A （normal control） and group B （severe scald, 30% TBSA, third-degree）. The rabbits were operated 24 hours after scald （group B） or fake scald （group A） and the peripheral blood was obthined from the carotid artery in order to separate PMNs. The PMNs in each sample were damaged and the total proteins of cells were extrac- ted. The proteins were separated by 2-DE using the immobilized pH gradient and SDS^PAGE. The gels were stained with coomassie brilliant blue and were scanned. The images were analyzed by PDquest software. The protein spots which were of discrepant expression were sieved and then analyzed by MALDI-TOF MS. The PMNs were obtained and were input into the data bank of proteins for identification of the proteins. [Results] Clear and high resolution 2-DE images of peripheral blood PMNs of rabbits were obtained. The amount of av- erage spots was 759±20 （group A） and 749 ±27 （group B）, respectively. The average matching rate was 93% （group A） and 91% （group B）, respectively. The 2-DE images of PMNs in group B differed from that of group A. Nineteen protein spots expressed discrepantly were sieved and their peptide mass fingerprints were obtained by MALDI TOF MS. Among the 19 protein spots, 8 spots （including 7 proteins） were identified by searching the data bank of proteins. The identified proteins included protein disulfide isomerase, beta-actin, annexin 1 and thiol specific antioxidant protein. [Conclusion] The proteome of peripheral blood PMNs of rab- bits alters in the condition of severe burns. The proteins expressed discrepantly include protein disulfide isomerase, beta-actin, annexin I and thiol-specific antioxidant protein, which are associated with enzyme activi- ty, moving function and flexibility, inflammation, metabolism and apoptosis of cells. These proteins are asso- ciated with the functional variation of PMNs in rabbits with severe burns.