摘要
目的 探讨严重烫伤大鼠延迟复苏后脾脏高迁移率族蛋白B1（HMGB1）表达对调节性T淋巴细胞（Treg）表型及其介导T淋巴细胞免疫功能的影响.方法 将104只Wistar大鼠按随机对照表法分为正常对照组8只、假烫组32只、烫伤组32只、丙酮酸乙酯（EP）治疗组32只.后2组大鼠造成30%TBSAⅢ度烫伤,伤后6 h腹腔注射林格液或EP液延迟抗休克,伤后12 h起每隔12 h给予4 mL林格液或EP液至伤后48 h;假烫组除于37℃模拟烫伤外其余处理同烫伤组.于伤后1、3、5、7 d处死各致伤组大鼠,另处死正常对照组大鼠,免疫磁珠法分离大鼠脾脏CD4~＋CD25~＋Treg,ELISA法检测脾脏HMGBI含量及T淋巴细胞上清液中IL-2水平,采用流式细胞仪检测Treg表面分子细胞毒性T淋巴细胞相关抗原4（CTLA-4）表达,同时检测T淋巴细胞增殖活性（数据以吸光度值表示）.对数据进行多组间方差分析和2组独立样本间的t检验.结果 （1）烫伤组大鼠脾脏HMGBI含量伤后1～7 d显著高于假烫组,其中第1天达峰值[（46.7±8.3）ng/mg蛋白];EP治疗组大鼠脾脏HMGBI含量伤后1～7 d明显低于烫伤组.（2）与假烫组比较,烫伤组大鼠伤后1～5 d CTLA-4表达明显增强（t值分别为10.459、12.051、4.029,P〈0.05或P〈0.01）;EP治疗组伤后1～7 d CTLA-4表达较烫伤组显著降低（t值分别为2.796、9.913、9.581、10.022,P〈0.05或P〈0.01）.（3）与假烫组比较,烫伤组大鼠伤后1～7 d脾脏T淋巴细胞增殖活性明显受抑,其中伤后1 d达低谷（0.167±0.059）;IL-2水平伤后1～7 d显著下降,其中伤后5 d达低谷（44±24）pg/mL.与烫伤组比较,EP治疗组伤后各时相点脾脏T淋巴细胞增殖受抑状态明显缓解,且IL-2水平明显上升.结论 严重烫伤后HMGB1产生可诱导Treg向成熟发展,从而介导T淋巴细胞增殖反应低下,免疫功能抑制.EP通过抑制HMGB1的合成与释放,改善烫伤延迟复苏大鼠的细胞免疫功能紊乱.
Objective To observe the influence of high mobility group box-1 protein （ HMGB1） derived from spleen on the phenotype of regulatory T lymphocytes （Treg） and HMGB1-mediated immune function in severely scalded rats after delayed resuscitation. Methods One hundred and four Wistar rats were divided into normal control group （NC, n =8） , sham scald group （SS, n =32） , scald group （S, n =32） , and ethyl pyruvate （ EP） treatment group （ EPT, n =32） according to the random comparison table. Rats in the latter 2 groups were subjected to 30% TBSA full-thickness scald, which were intraperitoneally injected with Ringer solution or EP solution at post scald hour （PSH） 6 （delayed antishock treatment） and administered with 4 mL Ringer solution or EP solution per 12 hours after PSH 12 till PSH 48. Rats in SS group were treated the same as that of S group except for sham scald with 37 ℃ water. Injured rats were sacrificed at post scald day （ PSD） 1 , 3 , 5 , 7 （ rats in NC group were also sacrificed） , and CD4^＋ CD25^＋ Treg were isolated from spleen with magnetic-activated cell sorting method. The content of HMGB1 in spleen and IL-2 level in supernatant were determined with ELISA. The expression of cytotoxic T lymphocyte-associated antigen 4 （CTLA-4） on Treg was determined with flow cytometry, and the proliferation activity of T lymphocytes was also detected （recorded as absorbance value）. Data were processed with analysis of variance among groups and independent samples t test. Results （ 1 ） Compared with that of rats in SS group and EPT group, the expression of splenic HMGB1 in S group increased significantly on PSD 1 through PSD 7 [peaked on PSD 1： （46.7 ±8.3） ng/mg protein]. （2） Compared with that in SS group, the expression of CTLA-4 in S group was enhanced significantly on PSD 1 through PSD 5 （ with t value respectively 10. 459, 12. 051 , 4. 029, P 〈 0.05 or P 〈0.01） ; while that in EPT group decreased significantly on PSD 1 through PSD 7 as compared with that from S group （with t value respectively 2. 796, 9. 913, 9.581, 10.022, P 〈 0.05 or P 〈0.01）. （3） Compared with that of rats in SS group, the proliferation activity of T lymphocytes in S group was markedly suppressed on PSD 1 through PSD 7 （ nadir on PSD1： 0. 167 ±0. 059） , and release of IL-2 was decreased significantly [nadir on PSD 5： （44 ±24） pg/mL]. T lymphocytes proliferation activity was restored and excretion of IL-2 increased in EPT group as compared respectively with that of S group at each time point. Conclusions The release of HMGB1 may stimulate splenic Treg to mature, thereby induce suppression of proliferation activity of T lymphocytes and immune function. EP can ameliorate immune dysfunction in animals with delayed resuscitation through inhibiting the synthesis and release of HMGB1.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2010年第2期104-108,共5页
Chinese Journal of Burns
基金
国家重点基础研究发展计划（2005CB522602）
国家自然科学基金（30872683、30901561）
