高迁移率族蛋白B1对严重烧伤大鼠枯否细胞的影响及晚期糖基化终末产物受体的作用  

Effectof high mobility group box protein 1 on the Kupffer cells of rats with severe burn and the role of receptor for advanced glycation end products in the process

作　　者：孙立[1] 陈旭林[1] 郭峰[1] 王飞[1] 刘晟[1] 梁勋[1] 王仁素[1] 王永杰[1] 孙业祥[1] 

机构地区：[1]安徽医科大学第一附属医院烧伤科,安徽合肥230022

出　　处：《中华烧伤杂志》2013年第2期158-161,共4页Chinese Journal of Burns

基　　金：国家自然科学基金（30872687、81000836）

摘　　要：目的 探讨高迁移率族蛋白B1（HMGB1）对严重烧伤大鼠枯否细胞（KC）分泌促炎性细胞因子的影响及晚期糖基化终末产物受体（RAGE）在该过程中的作用。方法 将32只SD大鼠背部浸于98 ℃水中12 s，制成30%TBSA Ⅲ度烫伤（以下称烧伤）。伤后24 h，处死大鼠分离肝脏KC（32个样本），以每孔1×106个接种至24孔板中。（1）取部分细胞按随机数字表法分为2组，每组样本数为8。对照组，加入1 mL PBS液培养；HMGB1组，加入1 mL 浓度为100 ng/mL HMGB1刺激。培养48 h后，采用蛋白质印迹法检测细胞表面RAGE的表达（结果以灰度值比值表示）。（2）取部分细胞采用随机数字表法分为4组，每组样本数为8。对照组，加入1 mL PBS液培养；HMGB1组，加入1 mL 浓度为100 ng/mL HMGB1刺激；HMGB1＋抗RAGE抗体组，经1 mL 浓度为20 μg/mL抗大鼠RAGE单克隆抗体孵育2 h，加入1 mL浓度为100 ng/mL HMGB1刺激；HMGB1＋重组大鼠RAGE/Fc 嵌合体（rrRAGE/Fc）组，将0.5 mL浓度为100 ng/mL HMGB1 与0.5 mL浓度为5 μg/mL rrRAGE/Fc孵育2 h后，作用于细胞。培养48 h后，采用ELISA法检测细胞培养上清液中TNF-α 和IL-1β的含量，RNA印迹法检测细胞内TNF-α和IL-1β mRNA的表达水平（结果以灰度值比值表示）。对数据进行单因素方差分析、t检验及LSD检验。结果 （1）培养48 h后，HMGB1组细胞RAGE表达水平（1.036±0.101）明显高于对照组（0.191±0.024，t=-23.158，P=0.000）。（2）HMGB1组、HMGB1＋抗RAGE抗体组以及HMGB1＋rrRAGE/Fc组细胞培养上清液中TNF-α含量分别为（10.59±1.39）、（9.91±1.68）、（11.51±2.27）ng/mL，IL-1β含量分别为（2.49±0.33）、（2.08±0.32）、（2.42±0.42）ng/mL；细胞内TNF-α mRNA水平分别为0.311±0.009、0.301±0.047、0.326±0.016，IL-1β mRNA水平分别为0.237±0.021、0.244±0.041、0.245±0.013，3组间比较差异均无统计学意义（P值均大于0.05），均显著高于对照组�Objective To investigate the effect of high mobility group box protein 1 （HMGB1） on the production of pro-inflammatory cytokines by Kupffer cell （KC） of rats with severe burn and the role of receptor for advanced glycation end products （RAGE） in the process.MethodsModel of 30% TBSA full-thickness burn was reproduced in 32 SD rats through immersing the back in 98 ℃ water for 12 s. KC （32 samples） was isolated from rat liver 24 h after injury and inoculated in 24-well plate in the concentration of 1×106 cell per well. （1） Cells were divided into control group （cultured with 1 mL PBS） and HMGB1 group （stimulated with 100 ng/mL HMGB1 in the volume of 1 mL） according to the random number table, with 8 samples in each group. At post culture hour （PCH） 48, the expression of RAGE （denoted as grey value ratio） was detected with Western blotting. （2） Another portion of cells were divided into control group （cultured with 1 mL PBS）, HMGB1 group （treated with 100 ng/mL HMGB1 in the volume of 1 mL）, HMGB1＋anti-RAGE antibody group （treated with 100 ng/mL HMGB1 in the volume of 1 mL after being pre-incubated with 20 μg/mL anti-RAGE monoclonal antibody in the volume of 1 mL for 2 hours）, HMGB1＋recombinant rat RAGE/Fc chimera （rrRAGE/Fc） group （treated with the mixture of 100 ng/mL HMGB1 in the volume of 0.5 mL and 5 μg/mL rrRAGE/Fc in the volume of 0.5 mL which were pre-incubated for 2 hours） according to the random number table, with 8 samples in each group. At PCH 48, the protein levels of TNF-α and IL-1β in supernatant were determined with enzyme-linked immunosorbent assay, while the mRNA expression of TNF-α and IL-1β （denoted as grey value ratio） were determined with Northern blotting. Data were processed with one-way analysis of variance, t test, and LSD test.Results（1） The expression of RAGE in HMGB1 group （1.036±0.101） was significantly higher than that of control group at PCH 48 （0.191±0.024, t=-23.158, P=0.000）. （2） In HMGB1 g

关 键 词：烧伤 枯否细胞 高迁移率族蛋白质类 晚期糖基化终末产物受体 促炎性细胞因子 

分 类 号：R6[医药卫生—外科学]