自噬对脓毒症小鼠中性粒细胞程序性死亡配体-1表达的影响  

Effect of autophagy on expression of neutrophil programmed death ligand-1 in mice with sepsis

作　　者：黄佳敏 孙燃 戚欣欣 刘璐 杨云稀 孙炳伟 Huang Jiamin;Sun Ran;Qi Xinxin;Liu Lu;Yang Yunxi;Sun Bingwei(Department of Burns and Plastic Surgery,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,Jiangsu,China;Department of Burn and Plastic Surgery,Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou 215002,Jiangsu,China)

机构地区：[1]江苏大学附属医院烧伤整形科,镇江212001 [2]南京医科大学附属苏州医院烧伤整形科,215002

出　　处：《中华危重病急救医学》2019年第9期1091-1096,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81471903,81772135);江苏省科技项目(BE2017695)。

摘　　要：目的探讨自噬对脓毒症小鼠中性粒细胞程序性死亡配体-1(PD-L1)表达的影响及其作用机制。方法①体内实验:将6~8周龄雄性C57BL/6小鼠按随机数字表法分为假手术组(Sham组)、盲肠结扎穿孔术(CLP)组和雷帕霉素(RAP)+CLP组,每组10只。采用CLP模拟脓毒症模型;Sham组不结扎盲肠及穿孔,其他操作同CLP组。RAP+CLP组小鼠于制模前7 d腹腔注射自噬激动剂RAP 4 mg·kg-1·d-1;Sham组和CLP组不予任何处理。各组于术后4 d留取肺脏、肝脏、脾脏和胰腺组织进行免疫组化染色,光镜下观察各器官中性粒细胞的浸润情况;通过免疫荧光染色检测术后1 d、4 d小鼠肺脏中性粒细胞免疫抑制分子PD-L1和自噬标志蛋白微管相关蛋白1轻链3(LC3)的表达。②体外实验:提取和重悬小鼠骨髓中性粒细胞至1×1010/L,分为空白对照组(不给予任何处理)、RAP对照组(RAP 100μmol/L)、自噬抑制剂Bafilomycin A1(Baf)对照组(Baf 10μmol/L)、脂多糖(LPS)刺激组(LPS 1 mg/L)、RAP+LPS组、Baf+LPS组,后两组分别于LPS刺激前30 min给予100μmol/L RAP或10μmol/L Baf预处理。于LPS刺激0、4、12 h用反转录-聚合酶链反应(RT-PCR)检测中性粒细胞PD-L1 mRNA表达;采用蛋白质免疫印迹试验(Western Blot)检测PD-L1、LC3和自噬相关蛋白p62的蛋白表达。结果①体内实验:免疫组化显示CLP术后4 d小鼠肺脏、肝脏、脾脏和胰腺组织中均有大量中性粒细胞浸润;免疫荧光显示,随CLP术后时间延长,小鼠肺脏中性粒细胞LC3阳性表达呈降低趋势,而PD-L1的表达大幅度升高;给予RAP预处理可以促进CLP小鼠LC3的表达,同时抑制PD-L1的表达。②体外实验:在mRNA水平上,随LPS刺激时间延长,小鼠中性粒细胞PD-L1 mRNA表达均呈持续升高趋势,12 h达峰值,且明显高于空白对照组(2-ΔΔCT:72.2±10.0比13.0±0.8,P<0.01);给予自噬抑制剂RAP预处理后,PD-L1的mRNA表达较LPS刺激组明显降低(2-ΔΔCT:12 h为47.4±7.3比72.2±10.0,P<0.01);而�Objective To investigate the effect and mechanism of autophagy on the expression of neutrophil programmed death ligand-1(PD-L1)in mice with sepsis.Methods①In vivo experiment:male C57BL/6 mice aged 6-8 weeks were divided into sham operation group(Sham group),cecum ligation and perforation(CLP)group,and rapamycin(RAP)+CLP group by random number table with 10 mice in each group.The sepsis model was reproduced by CLP,and the cecum and perforation were not ligated in Sham group,and other operations were the same as CLP group.The mice in RAP+CLP group were intraperitoneally injected with autophagy agonist RAP 4 mg·kg-1·d-17 days before modeling,while the mice in Sham group and CLP group were not treated.Lung,liver,spleen and pancreas tissues were harvested for immunohistochemical staining 4 days after the operation,and the infiltration of neutrophils in various organs was observed under light microscope.Meanwhile,the expressions of immunosuppressive molecule PD-L1 and autophagy marker microtubule-associated protein 1 light chain 3(LC3)in lung neutrophils were determined by immunofluorescence staining.②In vitro experiment:mouse bone marrow neutrophils were extracted and re-suspended to 1×1010/L,and they were divided into blank control group(without any treatment),RAP control group(RAP 100μmol/L),autophagy inhibitor Bafilomycin A1(Baf)control group(Baf 10μmol/L),lipopolysaccharide(LPS)stimulation group(LPS 1 mg/L),RAP+LPS group,and Baf+LPS group.The latter two groups were pretreated with 100μmol/L RAP or 10μmol/L Baf 30 minutes before LPS stimulation,respectively.The expression of PD-L1 mRNA of neutrophils was determined by reverse transcription-polymerase chain reaction(RT-PCR)at 0,4,12 hours after LPS stimulation.At the same time,the expressions of PD-L1,LC3 and p62 at the protein level were determined by Western Blot.Results①In vivo experiment:according to immunohistochemical experiments,a large amount of infiltration of neutrophils in lung,liver,spleen and pancreas was found at 4 hours after CLP.In the i

关 键 词：脓毒症 中性粒细胞 自噬 程序性死亡配体-1 雷帕霉素 

分 类 号：R73[医药卫生—肿瘤]