摘要
目的分析脓毒症模型小鼠腹腔巨噬细胞自噬性能的波动情况。方法生存实验小鼠随机分为WT/LPS组(n=12)以及IRF-1 KO型LPS组(n=12)。向LPS组小鼠腹腔中输入180~230μl LPS(20 mg/kg)。完成LPS输入后的96 h后采集小鼠的生存曲线。其余在体动物实验小鼠分为WT/NS组、WT/LPS组、IRF-1 KO/NS组、IRF-1 KO/LPS组,向LPS组小鼠腹腔中输入180~230μl 20 mg/kg的LPS,向NS组小鼠腹腔输入等量的LPS(20 mg/kg),完成注射12 h后全部处死,采集小鼠肺组织标本和肺泡巨噬细胞、脾脏巨噬细胞及腹腔巨噬细胞。利用免疫荧光染色法对各组小鼠腹腔巨噬细胞中的LC3B颗粒聚集情况进行检测,通过Western blot检测各组小鼠巨噬细胞cleaved caspase-3,LC3-Ⅱ/Ⅰ的表达自噬水平。结果 WT/LPS组的病理组织学出现急性肺损伤的情况,IRF-1 KO/LPS组的急性肺损伤的情况有所减缓;WT/LPS组的小鼠腹腔巨噬细胞以及脾脏巨噬细胞中的cleaved caspase-3蛋白表达水平、LC3-Ⅱ蛋白表达水平、LC3-Ⅱ/Ⅰ的比例均要高于WT/NS组,IRF-1 KO/LPS组的小鼠腹腔巨噬细胞中的cleaved caspase-3蛋白表达水平要低于WT/LPS组,该组LC3-Ⅱ蛋白表达水平、LC3-Ⅱ/Ⅰ的比例、LC3-Ⅰ向LC3-Ⅱ的转化均高于WT/LPS组;通过LPS诱导,WT组小鼠腹腔巨噬细胞线粒体损伤较为严重,IRF-1 KO组小鼠腹腔巨噬细胞产生线粒体自噬的情况;WT/LPS组以及IRF-1 KO/LPS组小鼠腹腔巨噬细胞中的LC3Bd颗粒聚焦程度,分别高于WT/NS组和WT/LPS组,IRF-1 KO组小鼠腹腔巨噬细胞自噬程度比WT组大。结论 IRF-1 KO对脓毒症的预后及脏器炎症有明显的修复作用,LRF-1干扰了小鼠巨噬细胞的凋亡和自噬平衡。
This study was designed to investigate the autophagic properties of peritoneal maerophages in septic mice. The mice were recruited and randomly divided into WT/LPS group and IRF-1 KO LPS group, with 12 mice in each group. 180~230 p^l LPS （20 mg/kg） was injected into the abdominal cavity of LPS mice. After 96 hours of LPS input, the survival curve of mice was calculated. In animal experiment in vivo, mice were divided into WT/NS group, WT/LPS group, IRF-1 KO/NS group, and IRF-1 KO/LPS group, in which, WT/LPS group and IRF-1 KO/ LPS group mice were injected with 180-230 Ixl LPS （20 mg/kg）, while NS mice were intraperitoneally injected with the same dose of LPS. All mice were sacrificed at 16 h after the completion injection, and the lung tissue specimens and mouse alveolar macrophages, splenic macrophages and peritoneal macrophage were collected. Furthermore,LC3B particle aggregation in peritoneal macrophages were detected by immunofluorescenee staining, the expression levels of cleaved caspase-3 and LC3-Ⅱ/Ⅰ were detected by Western blotting. Mice in WT/LPS group showed a pathological appearance of acute lung injury, and this injury was alleviated in IRF-1 KO/LPS group; peritoneal macrophages and spleen macrophages of WT/LPS represented higher levels of cleaved caspase-3 protein, LC3-Ⅱ protein, and LC3-Ⅱ/ I rate, as compared with WT/NS group; peritoneal macrophages of IRF-1 KO/LPS demonstrated a lower level of cleaved caspase-3 protein, but higher levels LC3-Ⅱ protein, higher LC3-Ⅱ/Ⅰ rate, and higher conversion rate of LC3-Ⅰ to LC3-Ⅱ, as compared with the WT/LPS group. After LPS induction, peritoneal macrophage mitochondrial injury of WT group mice became more serious, and the peritoneal macrophage of IRF-1 KO group mice showed mitochondrial autophagy. The aggregation degree of LC3Bd particles in peritoneal macrophages of WT/LPS and IRF-1 KO/LPS groups were higher that in WT/NS and WT/LPS groups, respectively; the autophagy degree of peritoneal macrophages in IRF-1 KO group was higher than
出处
《免疫学杂志》
CAS
CSCD
北大核心
2018年第1期27-33,共7页
Immunological Journal
基金
福建省卫生计生委青年科研课题(2017-2-63)
关键词
脓毒症
小鼠腹腔巨噬细胞
自噬功能
动态变化
Sepsis
Mouse peritoneal macrophage
Autophagy function
Dynamic changes
