严重烧伤后小鼠腹腔巨噬细胞肿瘤坏死因子的变化及免疫功能机制(英文)

Changes and immune function mechanism of tumor necrosis factor-alpha in murine peritoneal macrophage after severe scald

背景:严重烫伤导致机体免疫系统各方面的功能紊乱,活化的巨噬细胞可分泌许多生物活性递质。烧伤后巨噬细胞功能紊乱与信号转导的关系目前尚不清楚。目的:观察严重烫伤后不同时相点及运用特异性NF-κB抑制剂吡咯啉烷二硫代氨基甲酸盐(pyrrolidinedithiocarbamate,PDTC)后小鼠腹腔巨噬细胞内NF-κB活性、IκB-α的表达及肿瘤坏死因子(TNF-α)的变化,从信号转导的角度探讨巨噬细胞功能紊乱的机制。设计:随机对照的实验研究。单位:创伤、烧伤与复合伤国家重点实验室。材料:实验于1999-01/06在第三军医大学烧伤研究所实验室(国家级)完成。实验动物为健康清洁级近交系昆明小白鼠30只。干预:以小鼠常规烫伤模型造成体表面积15%Ⅲ度烫伤。实验按烫伤前及伤后不同时相随机分为6组,即0(正常对照组),2,6,12,24,48h组。收集腹腔巨噬细胞。采用放射免疫法检测TNF-α的含量,电泳迁移率改变分析法测NF-κB的活性,免疫印迹法测IκB-α的表达,反转录-PCR测TNF-αmRNA的表达。主要观察指标:①检测TNF-α的含量。②测定NF-κB的活性。③检测IκB-α的表达。④测TNF-αmRNA的表达。结果:烫伤后巨噬细胞分泌TNF-α亢进,于伤后12h达到高峰,为(1085.65±122.99)ng/L,较正常对照组明显增高(t=14.92,P<0.01)。NF-κB活性于伤后明?

BACKGROUND:Severe scald injury leads to a variety of disorders in the immune system.Activated macrophages are known to secrete many kinds of biologically active transmitter,but the relation between the functional disorder of the macrophages and signal transduction after burn injury has not been fully understood. OBJECTIVE:To observe the changes in nuclear factor(NF) κ B activity and expressions of Iκ B α and tumor necrosis factor(TNF) α in peritoneal macrophage of mice at different time points after severe scald injury and after the application of specific NF κ B inhibitor pyrrolidine dithiocarbamate(PDTC),thereby to explore the mechanism of macrophage dysfunction in light of signal transduction. DESIGN:A randomized controlled experimental research. SETTING:State Key Laboratory of Trauma,Burn and Combined Injury. MATERIALS:The experiment was carried out in the State Key Laboratory of Institute of Burn Research,Third Military Medical University during the period from January to June,1999,using 30 healthy clean grade Kunming mice of inbred strain. INTERVENTIONS: Common scald injury models(with third degree burn of 15% total body surface area) were established in the mice,which were randomized into 6 groups according to different time points after the injury for observation,namely 0 hour(normal control group) and postburn 2,6,12,24 and 48 hours.Peritoneal macrophages were collected at these time points for examining TNF α content using radio immunoassay and NF κ B activity by means of electrophoretic mobility shift assay(EMSA).The expressions of Iκ B α and TNF α mRNA were determined by immunoblotting method and reverse transcription PCR,respectively. MAIN OUTCOME MEASURES:Examinations of ① the content of TNF α , ② NF κ B activity,③ expression of Iκ B α , and ④ expression of TNF α mRNA. RESULTS:Macrophage secretion of TNF α was enhanced postburn,reaching the peak level at 12 hours[(1 085.65± 122.99)ng/L],which was significantly higher than that in the normal control group(t=14.92,P< 0.01). Postburn NF κ B activity significantly increased after the injury, peaking at 2 hours[(56.8± 7.3)RDU], which occurred much earlier than the peak of TNF α secretion(t=13.31,P< 0.01).Compared with that in the normal control group, Iκ B α expression decreased significantly 2 hours postburn(t=4.23, P< 0.01) to 0.632± 0.086,followed by gradual increase to the peak level to 1.161± 0.097 24 hours after the burn injury(t=7.06,P< 0.01) and then by slight decrease to 1.149± 0.167 till 48 hours(t =4.82,P< 0.01).Twelve hours after injury was the time point for intervention with PDTC application, when NF κ B activity and TNF α mRNA expression both decreased significantly(P< 0.01). CONCLUSION:NF κ B activity and TNF α mRNA expression decrease significantly after severe scald.At high levels,Iκ B α and NF κ B maintain an interaction for their restriction.After burn injury,NF κ B signal transduction pathway is involved in the modulation of TNF α expression in mouse peritoneal macrophage.