严重烫伤后小鼠腹腔巨噬细胞NF-κB、IκB-α、TNF-α的变化及调控

Changes and regulation of NF-κB activity and IκB-α as well as TNF-α expression of murine peritoneal macrophages following severe burns

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PDTC后小鼠腹腔巨噬细胞内NF κB活性、IκB α的表达及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。方法 以 1 5%体表面积Ⅲ度烫伤小鼠为模型 ,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 ,改良的ELISA及WesternBlotting法测IκB α的表达 ,反转录 PCR测TNF αmRNA的表达。结果 烫伤后巨噬细胞分泌TNF α亢进 ,于伤后 1 2h达到高峰。NF κB伤后明显活化 ,于 2h达到了高峰 ,早于TNF α的增多 ,IκB α的表达先降低 ,后升高。予PDTC后NF κB活性及TNF αmRNA表达量均下降。结论 烫伤后NF κB活性及TNF α表达明显增强 ,IκB α对NF κB在高水平上维持着一种制约关系。烫伤后小鼠腹腔巨噬细胞内NF κB途径参与TNF

Objective To explore some mechanisms of macrophage functional disorder at the level of nuclear transcription factors by observing the dynamic changes of NF κB activity and TNF α secretion as well as IκB α expression by murine peritoneal macrophages (PMΦs) at different time intervals and after using NF κB inhibitor PDTC. Methods Model of 15% TBSA fullthickness scalded mice was used as experimental animal and PMΦs were collected. The following parameters were measured with the method indicated in parathesis: TNF α in supernatant of cultured PMΦs(RIA), TNF α mRNA (RT PCR), NF κB activity (electrophoretic mobility shift assay, EMSA), IκB α expression (modified ELISA and Western blotting). Results NF κB activity and TNF α secretion were markedly activated and reached their peak 2 h after burn and 12 h after burn respectively. Expression of IκB α decreased firstly and then increased to high level. After using NF κB specific inhbitor PDTC, NF κB activity and TNF α mRNA expression decreased at the same time. Conclusions NF κB activity and TNF α secretion are markedly activated after severe scald. High IκB α expression exhibits a strong regulatory effect on NF κB activation of PMΦs. NF κB pathway participate in the regulation of TNF α expression of macrophage after severe scald.