摘要
目的探讨严重创伤后小鼠腹腔巨噬细胞(PM)中芳香烃受体(AhR)的表达规律并分析增强创伤后PM的AhR表达对炎症因子水平和杀菌能力的影响。方法采用实验研究方法。取40只6~8周龄雄性C57BL/6J小鼠(小鼠周龄、性别、品系下同),按随机数字表法(分组方法下同)分为对照组、创伤后2 h组、创伤后6 h组、创伤后12 h组,每组10只。将后3组小鼠构建骨折+失血的严重创伤模型,对照组小鼠不做处理。分别在未创伤和创伤后2、6、12 h时,提取对照组、创伤后2 h组、创伤后6 h组、创伤后12 h组小鼠原代PM(提取细胞下同),分别采用蛋白质印迹法和实时荧光定量反转录PCR法检测AhR的蛋白和mRNA表达,采用转录组测序分析AhR信号通路相关分子的基因表达。取20只小鼠分为对照组、创伤后6 h组,每组10只,提取PM后,采用免疫沉淀法检测AhR泛素化水平。取12只小鼠,分为单纯二甲基亚砜(DMSO)组、创伤后6 h+DMSO组、单纯MG-132组、创伤后6 h+MG-132组,每组3只,并行相应处理后,提取PM,采用蛋白质印迹法检测AhR蛋白的表达。取20只小鼠构建创伤后6 h模型并提取PM,分为空载腺病毒(Ad-NC)组和AhR过表达腺病毒(Ad-AhR)组,部分细胞转染相应腺病毒36 h后,采用蛋白质印迹法检测AhR的蛋白表达;将剩余Ad-NC组细胞分为单纯Ad-NC组、Ad-NC+内毒素/脂多糖(LPS)组,将剩余Ad-AhR组细胞分为单纯Ad-AhR组和Ad-AhR+LPS组,并行相应处理12 h后,采用酶联免疫吸附测定法检测细胞上清液中白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)的表达(样本数为6)。取20只小鼠提取PM并分为对照+Ad-NC组、创伤后6 h+Ad-NC组、对照+Ad-AhR组、创伤后6 h+Ad-AhR组,并行相应处理后,采用平板涂布法检测细胞内载菌量(样本数为6)。对数据行单因素方差分析、LSD检验、析因设计方差分析、独立样本t检验。结果与对照组(1.16±0.28)比较,创伤后2 h组(0.59±0.14)、创伤后6 h组(0.7
Objective To explore the expression pattern of aryl hydrocarbon receptor(AhR)in mice peritoneal macrophages(PMs)after major trauma and analyze the effects of enhanced AhR expression on the inflammatory cytokine level and bactericidal ability after trauma.Methods The experimental study method was used.Forty 6-8-week-old male C57BL/6J mice(the same mouse age,sex,and strain below)were divided into control group,post trauma hour(PTH)2 group,PTH 6 group,and PTH 12 group according to the random number table(the same grouping method below),with 10 mice in each group.Mice in the latter 3 groups were constructed as severe trauma model with fracture+blood loss,while mice in control group were left untreated.The primary PMs(the same cells below)were extracted from the mice in control group,PTH 2 group,PTH 6 group,and PTH 12 group when uninjured or at PTH 2,6,and 12,respectively.Then the protein and mRNA expressions of AhR were detected by Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction,respectively,and the gene expressions of AhR signaling pathway related molecules were analyzed by transcriptome sequencing.Twenty mice were divided into control group and PTH 6 group,with 10 mice in each group,and the PMs were extracted.The level of ubiquitin of AhR was detected by immunoprecipitation.Twelve mice were divided into dimethyl sulfoxide(DMSO)alone group,PTH 6+DMSO group,MG-132 alone group,and PTH 6+MG-132 group,with 3 mice in each group.After the corresponding treatment,PMs were extracted,and the protein expression of AhR was detected by Western blotting.Twenty mice were constructed as PTH 6 model.Then,the PMs were extracted and divided into empty negative control adenovirus(Ad-NC)group and AhR overexpression adenovirus(Ad-AhR)group.The protein expression of AhR was detected by Western blotting at 36 h after some PMs were transfected with the corresponding adenovirus.The rest cells in Ad-NC group were divided into Ad-NC alone group and Ad-NC+endotoxin/lipopolysaccharide(LPS
作者
旷田垠
殷双琴
代伟宏
罗莉
陈涛
梁星河
王日兴
梁华平
朱俊宇
Kuang Tianyin;Yin Shuangqin;Dai Weihong;Luo Li;Chen Tao;Liang Xinghe;Wang Rixing;Liang Huaping;Zhu Junyu(State Key Laboratory of Trauma,Burns and Combined Injury,Department of Wound Infection and Drug,Daping Hospital,Army Medical University,Chongqing 400042,China;Emergency Department of the Second Affiliated Hospital of Hainan Medical University,the Emergency and Critical Care Clinical Medicine Research Center of Hainan,Haikou 570216,China;The 17th Team of Cadet Brigade,College of Basic Medical Sciences,Army Medical University,Chongqing 400038,China)
出处
《中华烧伤与创面修复杂志》
CAS
CSCD
北大核心
2023年第7期633-640,共8页
Chinese Journal of Burns And Wounds
基金
重庆市自然科学基金面上项目(cstc2021jcyj-msxmX0234)
重庆市博士“直通车”科研项目(CSTB2022BSXM-JCX0009)
中央引导地方科技发展专项资金(ZY2021HN19)
国家级大学生创新创业训练计划(202190035010)。
关键词
巨噬细胞
创伤和损伤
芳香烃受体核转位子
炎症因子
杀菌能力
Macrophages
Wounds and injuries
Aryl hydrocarbon receptor nuclear translocator
Inflammatory cytokine
Bactericidal ability
