严重烫伤小鼠腹腔巨噬细胞肿瘤坏死因子α、蛋白激酶C的变化及调控

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.目的观察烧伤后不同时相点肿瘤坏死因子α(tumornecrosis factor-α,TNF-α)的变化及运用特异性调控剂H-7后小鼠腹腔巨噬细胞内核因子-κB(nuclearfactor-κB,NF-κB)活性及膜浆蛋白激酶C(proteinkinase C,PKC)的变化,在信号转导的水平探讨PKC是否参与了巨噬细胞TNF-α的调控,阐明巨噬细胞功能紊乱的某些机制.设计以实验动物为研究对象的随机对照的实验研究.单位一所军医大学的烧伤研究所.材料实验于1999-01/12在解放军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,32只.方法以本所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),2,6,12,24,48h6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,同位素掺入测膜浆PKC活性.主要观察指标①检测TNF-α的含量.②测定NF-κB的活性.③检测膜浆PKC的活性.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).与正常对照组相比,伤后膜浆PKC活性升高,其中膜PKC活性于伤后2h显著增高(t=7.930,P＜0.01),为(231.80±31.66)nmol/min·g.6h稍降,接近于正常(t=2.531,P＜0.05),为(174.29±16.80)nmol/min·g,12h及24h迅速上升,至48h达到峰值(t=29.42,28.03,30.19,P＜0.01),分别为(512.10±33.42),(454.70±21.06)及(530.49±28.54)nmol/min·g.TNF-α和膜PKC的变化进行相关分析表明,二者呈显著的正相关(r=0.7964,P＜0.05).EMSA图像显示,烫伤后NF-κB活性明显升高.以伤后12 h为调控点,经H-7作用后,NF-κB活性显著被抑制.结论烧伤后小鼠腹腔巨噬细胞TNF-α的分泌及PKC,NF-κB的活性明显被激活,PKC-NF-κB信号通路参与了TNF-α表达的调控,为烧伤过程中免疫功能的调整及康复干预提供了实验学数据.

Alternation and modulation of tumor necrosis factor-alpha and protein kinase C in celiac macrophage of mouse after serious scalding

BACKGROUND: Serious scalding leads to dysfunction of each aspect in immune system, and activated macrophage can secret many bioactive transmitters. The relationship between macrophage dysfunction and signal conduction after scalding is unclear at present.OBJECTIVE: To observe the alternation of tumor necrosis factor- alpha (TNF-α) at different time points after scalding and the activity of nuclear factor κB(NF-κB) and alternation of protein kinase C (PKC) after the application of specific modulator H-7 to explore whether PKC participates in the modulation of TNF-α in macrophage on signal conduction level for the clarification of some mechanisms of macrophage dysfunction.DESIGN: A randomized controlled experimental study by employing experimental animals as subjectsSETTING: An institute of burn research of a military medical university MATERIALS: The study was conducted in the Laboratory (state) of the Institute of Burn Research, Third Military Medical University of Chinese PLA between January and December 1999. Experimental animals were 32 healthy clean inbreeding Kunming white mice.METHODS: 15% Ⅲ scalding was created in mice for the establishment of routine scalding model. Mice were randomly divided into 6 groups according to different time points before or after scalding, I.e. 0(normal control group), 2, 6, 12, 24, or 48 hours group. Celiac macrophages were collected for the detection of TNF-α content by radioimmunoassay, NF-κB activity by electrophoretic mobility shift analysis (EMSA), and membrane or plasma PKC activity by isotope analysis.MAIN OUTCOME MEASURES: ① TNF-α content; ② NF-κB activity; ③Membrane or plasma PKC activity RESULTS: After scalding, macrophage excessively secreted TNF-α and reached its peak of (1 085.65 ± 122.99) ng/L at 12 hours, which was significantly higher than that of control group( t = 14.92, P ＜ 0.01 ).Compared with control group, membrane PKC activity increased after scalding, which significantly heightened to(231.80 ± 31.66) nmol/min · g at 2hours( t = 7. 930, P ＜ 0.01 ), slightly decreased to close to normal level of (174.29±16.80) nmol/min· gat 6hours(t=2.531, P ＜0.05), and rapidly elevated at 12 hours [512. 10 ±33.42) nmol/min · g] and 24 hours [ (454.70 ± 21.06) nmol/min · g] to reach its peak of(530.49 ± 28.54)nmol/min. G at 48 hours( t = 29.42, 28.03, 30. 19, P ＜ 0. 01 ). Correlation analysis of the alternation between TNF-α and membrane PKC indicated a significant positive correlation( r = 0. 796 4, P ＜ 0. 05) . As indicated by EMSA image, NF-κB activity significantly elevated after scalding. Twelve hours after scalding was set as modulation point, NF-κB activity was significantly inhibited by the application of H-7.CONCLUSION: The secretion of TNF-α and the activities of PKC and NF-κB are significantly activated in celiac macrophage after scalding, and PKC-NF-κB signal pathway participates in the modulation of TNF-α expression, which provide experimental data for the modulation of immune function and rehabilitative intervention during scalding.serious scalding.