急性坏死性胰腺炎大鼠外周血中性粒细胞凋亡相关的蛋白质变化

Changes of neutrophil apoptosis-related proteins in peripheral blood of rats with acute necrotizing pancreatitis

目的观察急性坏死性胰腺炎（ANP）大鼠外周血中性粒细胞（PMN）蛋白组学的变化。方法SD大鼠60只，随机分为2组，每组30只，建立ANP组和SO（假手术）组模型，分别在制模后3、6、12h分批处死大鼠，抽取下腔静脉血测定血淀粉酶含量，密度梯度离心分离PMN，用流式细胞仪测定PMN凋亡比率。切取胰腺标本组织，病理切片苏木素-伊红（HE）染色，并对胰腺组织进行病理评分。同时对大鼠制模后12h的PMN裂解，应用Label free技术对两组PMN进行蛋白定量差异分析，DeCyderM STM2．0分析定量数据，MASCOT算法鉴定定量多肽分子，且进一步鉴定出与凋亡有关的差异蛋白质。结果ANP组PMN凋亡延迟，至12h达到最低（2．15±0．45），与SO组各个时间点比较差异有统计学意义（P〈0．01）；鉴定出与PMN凋亡有关的4种蛋白质相对分子质量为78000葡萄糖调节蛋白、RhoGTPases、L-乳酸脱氢酶A链和血红蛋白α2链（ANP组与SO组的比值分别为1．953614、3．526625、1．766764、0．609825），两组差异有统计学意义（P〈0．05）。结论ANP时，PMN凋亡延迟，与凋亡有关的差异蛋白质可能参与了ANP外周血PMN凋亡延迟。

Objective To observe the proteomic changes of peripheral blood neutrophils （PMN） in rats with acute necrotizing pancreatitis （ANP）. Methods Sixty SD rats were randomly divided into two groups （n =30 each） ： ANP group and sham-operated （SO） group. The rats were sacrificed at 3rd, 6th, and 12 h. The venous blood serum was taken from the inferior vena for determination of amylase content. Density gradient centrifugation was used to isolatie PMN. Apoptosis ratio of PMN was detected by using flow cytometry. The pathological sections from the excised pancreas tissue samples were subjected to hematoxylin and eosin （HE） staining and pathologically scored. PMN lysis molding in rats after 12 h at the same time, the application of Label free two groups of PMN the protein quantitative differences analysis using DeCyder MSTM 2. 0 analyze quantitative data, identification with MASCOT algorithm quantitative pol- ypeptide molecules, and further identified with apoptosis related differences in protein. Results ANP group PMN apoptosis delay, the lowest （2. 15 ± 0. 45） to 12 h, the difference was statistically significant （P 〈 0. 01 ） compared with the SO group at each time point ;identified four PMN apoptosis protein： 78 000 glucose regulatory proteins, the ratio of the the RhoGTPases, L-lactate dehydrogenase A chain and hemoglobin α2 chain （ ANP group and SO group, respectively 1. 953 614, 3. 526 625, 1. 766 764, 0. 609 825）, the difference was statistically significant （P 〈 0. 05 ）. Conclusion ANP, PMN apoptosis delay apoptosis-related differences in the protein may be involved in the ANP peripheral blood PMN apoptosis delay.