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目的研究重度烧伤大鼠血清刺激后巨噬细胞Notch1蛋白表达变化,及其细胞分泌因子白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的水平变化。方法选取成年雄性SD大鼠24只,随机分为假伤组、烧伤24 h组和烧伤7 d组,每组8只。烧伤组SD大鼠造成约30%总体表面积(TBSA)Ⅲ度烧伤,分别于伤后24 h、7 d收集血清;假伤组大鼠用37℃水浴,水浴后24 h取其血清作为对照。用上述各组血清制成20%培养液,刺激小鼠源巨噬细胞系RAW264.7,分别于加入血清刺激后即刻和刺激4、8、12、24、48 h后收取细胞及其上清液。另外用含20%假伤血清+脂多糖(100 ng/m L)培养液刺激巨噬细胞,于相同时间点收样。Western Blot检测各时间点巨噬细胞中Notch1蛋白表达变化,酶联免疫吸咐试验检测刺激24 h后培养上清液中IL-6及TNF-α的含量变化。结果 (1)假伤组血清刺激后即刻和刺激4、8、12、24、48 h后,Notch1蛋白表达无明显变化;(2)烧伤24 h组血清刺激后,Notch1蛋白表达明显升高,并随时间延长而达高峰;(3)烧伤7 d组血清刺激后,Notch1蛋白表达无明显变化,与假伤组血清刺激结果类似;(4)假伤组血清+脂多糖混合刺激后,Notch1蛋白表达明显增加;(5)烧伤24 h组血清及假伤组血清+脂多糖,上清液中IL-6和TNF-α含量均明显高于假伤组血清及烧伤7 d组血清。结论烧伤血清刺激下的巨噬细胞Notch1信号被激活,IL-6及TNF-α分泌能力增强,且这种激活可被脂多糖刺激所模拟。
Objective To investigate the changes of Notch1 protein expression in macrophages and the changes of the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in sera of severe burn rats. Methods Twenty-four adult male Sprague-Dawley rats were randomly divided into sham-injury group, 24-h burn group and 7-day burn group, with 8 rats in each group. Serum was collected at 24 h and 7 d after injury in SD rats in burn group, and the serum was taken as control at 37 ℃ for 24 h after water immersion. 20% of the above-mentioned serum was used to stimulate the mouse macrophage cell line RAW264.7, respectively, after the addition of serum stimulation and stimulation 4,8,12,24,48 h after harvest cells and supernatant liquid. In addition, macrophages were stimulated with 20% sham serum + lipopolysaccharide (100 ng / mL) medium at the same time point. The changes of Notch1 protein expression in macrophages were detected by Western Blot. The contents of IL-6 and TNF-α in culture supernatants were detected by enzyme-linked immunosorbent assay after 24 h of stimulation. Results (1) The expression of Notch1 protein was not changed immediately after stimulation of serum in sham injury group and 4, 8, 12, 24 and 48 h after stimulation; (2) Notch1 protein expression was significantly increased after 24 h of sepsis treatment, And reached the peak with the prolongation of time. (3) After stimulated with serum for 7 d, the expression of Notch1 protein did not change significantly, which was similar to that of sham group. (4) Notch1 (5) The contents of IL-6 and TNF-αin serum + lipopolysaccharide and supernatant of 24 h burn group were significantly higher than that of sham group and 7 d burn group. Conclusion The Notch1 signal of macrophages stimulated by burn serum is enhanced, and the secretion of IL-6 and TNF-α is enhanced. This activation can be simulated by stimulation of lipopolysaccharide.