重度烧伤大鼠血清对巨噬细胞Notch1的激活及分泌功能的影响

Effect of the activation and secretion function of macrophage Notch1 after the stimulation of severe burn rat serum

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 dynamic expression of Notch1 signaling and the secretion of interleukin-6( IL-6) and tumor necrosis factor alpha( TNF-α) from macrophage RAW264. 7 cells induced by severe burn rat serum. Methods Twenty-four male Sprague-Dawley rats were divided into sham burn group and burn at 24 h and 7 d groups randomly,each group had eight rats. The rats of burn groups were made the third degree burns about 30% total body surface area. The serum of burn rats were collected at24 h and 7 d after burn. The sham burn group was employed 37℃ water-bath and the serum was treated as a control. The mixed culture medium contained 20% burned serum,RAW264. 7 cells were cultivated with each group of burned serum for immediate moment,4,8,12,24,48 h. In addition,100 ng / m L lipopolysaccharide + 20% sham burn serum were applied to culture medium,which were employed to cultivate RAW 264. 7. The Notch1 signaling of macrophages were detected after cultivation and the secretion of IL-6 and TNF-α in culture supernatants of each group was measured by enzyme linked immunosorbent assay. Results( 1) The expression of Notch1 protein had no obvious change at immediate moment,4,8,12,24,48 h after stimulation of sham burn serum.( 2) In burn at 24 h group,the expression of Notch 1protein was significantly increased and as time progresses the signaling peaked.( 3) In burn at 7 d group,the expression of Notch 1 protein had no obvious change,which was flat as the sham burn serum group.( 4)In the mixed culture medium of lipopolysaccharide + sham burn serum group,the level of Notch1 signaling was significantly increased after stimulation.( 5) The contents of IL-6 and TNF-α in burn at 24 h group and in lipopolysaccharide + sham burn serum group were significantly higher than those of the sham burn serum group and the burn at 7 d group. Conclusion The Notch1 signaling and the secretion of IL-6 and TNF-α of macrophage could be stimulated by burn serum and this kind of high level signaling could be imitated by lipopolysaccharide.