【作者】 刘丽；

【导师】 史丽云；

【作者基本信息】 南京中医药大学 ， 免疫学， 2024， 硕士

【摘要】 背景:脓毒症是一种严重感染引起的全身性炎症性疾病,可累及多个全身器官,其中以肺最为易感,患者极易发生急性肺损伤(acute lunginjury,ALI)。有关脓毒症引起ALI的详细机制尚不完全清楚。脓毒症如得不到及时控制,会引起多脏器功能障碍,甚至导致死亡。在引发脓毒症的感染源中,革兰氏阴性菌是最常见的一种,而革兰氏阴性菌分泌的外膜囊泡(Outer membrane vesicles,OMVs)是直径为20～400 nm纳米样颗粒,具有双层膜结构。OMVs包括与致病相关的多种成分,如非蛋白抗原脂多糖、脂质、蛋白质粘附素、侵袭素、毒素等致病因子。OMVs还含有细菌的DNA、RNA、sRNA和质粒等核酸,与致病性、耐药基因的存储和传播、细菌间及细菌-宿主间通信串扰等一系列功能紧密相关。本实验室在之前的研究中发现在脓毒症小鼠中,大肠杆菌可能通过受损的肠屏障进入肺组织并促进肺损伤。然而大肠杆菌产生的细菌外膜囊泡在脓毒症中的具体作用机制尚不清楚。目的:探索大肠杆菌来源的细胞外囊泡在脓毒症诱导的肺损伤中的潜在作用机制。方法:通过差速离心法提取大肠杆菌来源的外膜囊泡,将荧光染料Dil标记的大肠杆菌来源的外膜囊泡通过灌肠的方式给予脓毒症小鼠后,记录造模后小鼠饮食,饮水,体温和体重的变化,并通过免疫荧光检测小鼠肺组织冰冻切片中Dil的荧光强度进行判断大肠杆菌来源的外膜囊泡是否可以通过受损的肠道转移至肺部。随后再将大肠杆菌来源的外膜囊泡用气道灌注的方式给予小鼠,通过H&E染色分析肺组织病理切片是否有炎症,细胞计数法和BCA法检测肺泡灌洗液中总细胞数和总蛋白的含量,流式细胞术检测免疫细胞(肺泡巨噬细胞,单核巨噬细胞,中性粒细胞)的比例变化等实验来分析大肠杆菌来源的外膜囊泡是否能够导致肺部炎症。同时通过肺组织免疫荧光检测Dil标记的大肠杆菌来源的外膜囊泡与肺部免疫细胞的共定位情况。在细胞水平和动物水平分别探索潜在机制。将肠杆菌来源的外膜囊泡处理肺泡巨噬细胞,通过qPCR、WB、肺组织免疫荧光等实验进一步探索诱导炎症的作用机制;将大肠杆菌来源的外膜囊泡和去除了 DNA的大肠杆菌外膜囊泡给予Stinggt/gt小鼠和WT小鼠,进一步分析大肠杆菌来源的外膜囊泡在脓毒症肺损伤中的潜在机制。结果:脓毒症小鼠在经过Dil标记的大肠杆菌来源的外膜囊泡灌肠处理之后,饮食饮水减少,体温体重降低,肺组织冰冻切片可见明显的Dil红色荧光,结果提示大肠杆菌来源的外膜囊泡可以通过脓毒症小鼠受损的肠屏障到达肺组织;大肠杆菌来源的外膜囊泡经气道灌注后,小鼠肺组织病理切片H&E染色表示,肺部炎性细胞增多,肺泡间隔增宽,肺泡灌洗液中总细胞数和总蛋白含量增加,中性粒细胞比例明显增多,肺泡巨噬细胞比例明显降低,单核巨噬细胞无明显变化,免疫荧光显示肺泡巨噬细胞共定位。结果显示,大肠杆菌来源的外膜囊泡能够诱导小鼠肺组织炎症,并显著性的影响降低肺泡巨噬细胞比例,升高中性粒细胞比例;肺泡巨噬细胞经过大肠杆菌来源的外膜囊泡处理后,由qPCR结果显示炎症因子和IFN-I的表达升高,WB结果提示cGAS-STING信号通路相关蛋白被激活;WT小鼠和Stinggt/gt小鼠在经过大肠杆菌来源的外膜囊泡或去除了 DNA的大肠杆菌来源的外膜囊泡处理后,H&E染色和免疫荧光结果提示,大肠杆菌来源的外膜囊泡能够导致导致WT小鼠肺部炎症。但WT或Sting gt/gt小鼠在经过E.coli OMVs(预加DNase处理)后,肺组织无显著炎症。在阻断IFN-I信号通路后,H&E染色显示,肺部炎症降低,免疫荧光结果提示,中性粒细胞比例升高。以上结果提示大肠杆菌来源的外膜囊泡可能是通过其DNA激活cGAS-STING信号通路诱导IFN-I促进脓毒症小鼠肺部炎症。结论:大肠杆菌来源的外膜囊泡可以通过脓毒症小鼠受损的肠屏障从肠转移至肺部,其机制可能是通过大肠杆菌来源的外膜囊泡的DNA激活肺泡巨噬细胞的cGAS-STING信号通路介导的。更多还原

【Abstract】 Backgrounds:Sepsis is a severe infection-induced systemic inflammatory disease that can involve multiple systemic organs,of which the lungs are the most susceptible,and patients are highly susceptible to acute lung injury(ALI).The mechanism of sepsis-induced ALI has not been fully understood.Uncontrolled sepsis can cause multiple organ failure and even death.Gram-negative bacteria are the most common infectious agents that cause sepsis,and Gram-negative bacteria secrete outer membrane vesicles(OMVs),which are nanoparticles with a diameter of 20-400 nm and a doublemembrane structure.OM Vs consist of a variety of components related to pathogenicity,such as non-protein antigenic lipopolysaccharides,lipids,proteins,proteins and toxins,and other pathogenic components.OMVs also contain bacterial nucleic acids such as DNA,RNA,sRNA and plasmids,which are closely related to pathogenicity,storage and transmission of drug resistance genes,and inter-bacterial and inter-host communication crosstalk.In previous studies in our laboratory,it was found that in septic mice,E.coli may enter lung tissue through the compromised intestinal barrier and promote lung injury.However the exact mechanism of action of bacterial outer membrane vesicles produced by E.coli in sepsis is unknown.Aim:Exploring the potential mechanism of action of extracellular vesicles of Escherichia coli origin in sepsis-induced lung injury.Methods:E.coli-derived outer membrane vesicles were extracted by differential centrifugation,and the fluorescent dye Dil-labelled E.coli-derived outer membrane vesicles were given to septic mice by enema.The changes in diet,water intake,body temperature and body weight of the mice after modelling were recorded,and the fluorescence intensity of Dil in frozen sections of mouse lung tissues was measured by immunofluorescence to determine whether E.coli-derived outer membrane vesicles could be transferred to the lungs via the damaged intestine.damaged intestines to the lungs.The E.coli-derived outer membrane vesicles were then given to the mice by airway perfusion,and H&E staining was used to analyse whether there was inflammation in the histopathological sections of lungs,cell counting and BCA to detect the total cell count and total protein content in the alveolar lavage fluid,and flow cytometry to detect the changes in the proportion of immune cells(alveolar macrophages,monocyte macrophages,neutrophils)to analyse whether the E.coli-derived outer membrane vesicles could cause lung inflammation.Whether the outer membrane vesicles were able to cause lung inflammation.The co-localization of Dil-labelled E.coli-derived outer membrane vesicles with lung immune cells was also examined by immunofluorescence in lung tissue.Potential mechanisms were explored at the cellular level and animal level,respectively.The E.coli-derived outer membrane vesicles were treated with alveolar macrophages to further explore the mechanism of inflammation induction by qPCR,WB lung tissue immunofluorescence,etc.E.coli-derived outer membrane vesicles and DNA-removed E.coli-derived outer membrane vesicles were given to Stingt/gt mice and WT mice,to further analyze the potential mechanism of E.coli-derived outer membrane vesicles in septic lung injury.Mechanisms.Results:After airway perfusion of E.coli-derived outer membrane vesicles,H&E staining of lung histopathological sections of mice indicated an increase in inflammatory cells in the lungs,widening of alveolar intervals,an increase in the total cell count and total protein content of alveolar lavage fluid,a significant increase in the proportion of neutrophils,a significant decrease in the proportion of alveolar macrophages,and no significant changes in mononuclear macrophages;septic mice after treatment with Dil-labelled E.coli-derived outer membrane vesicles In septic mice,after enema treatment with Dil-labelled E.coli-derived outer membrane vesicles,there was a decrease in diet and water intake,a decrease in body temperature and body weight,and a clear red fluorescence of Dil in the frozen sections of lung tissues;in alveolar macrophages after treatment with E.coli-derived outer membrane vesicles,the WB results indicated that E.coli-derived outer membrane vesicles may activate the inflammation through its DNA by activating its cGAS-STING signaling pathway;in Stinggt/gt and In Stinggt/gt mice and WT mice,after treatment with E.coli-derived outer membrane vesicles or DNA-removed E.coliderived outer membrane vesicles,H&E staining in Stinggt/gt mice showed that there was no obvious inflammation in the lung tissues,and the total cell counts and total protein contents of alveolar lavage fluid did not increase significantly,and the expression level of inflammatory factors detected by qPCR was also increased,which suggests that E.coli-derived outer membrane vesicles might cause inflammation through the cGAS-STING signaling pathway.This suggests that the E.coli-derived outer membrane vesicles may induce lung inflammation through the cGASSTING signalling pathway,whereas in WT mice,lung inflammation was reduced only after removing the DNA from the E.coli-derived outer membrane vesicles.After the septic mice were treated with Dil-labelled E.coli-derived outer membrane vesicles by enema,the diet and water intake were reduced,body temperature and body weight were lowered,and obvious Dil red fluorescence could be seen in frozen sections of lung tissues,which suggested that E.coli-derived outer membrane vesicles could reach the lung tissues through the damaged intestinal barrier of the septic mice;after E.coli-derived outer membrane vesicles were enucleated through the airway,the pathological sections of the lung tissues of the mice with H&E staining indicated an increase in inflammatory cells in the lungs,widening of alveolar septa,an increase in the total cell count and total protein content in the alveolar lavage fluid,a significant increase in the proportion of neutrophils,a significant decrease in the proportion of alveolar macrophages,and no significant changes in mononuclear macrophages,and immunofluorescence showed colocalization of alveolar macrophages.The results showed that E.coli-sourced outer membrane vesicles were able to induce inflammation in the lung tissues of mice and significantly The results showed that alveolar macrophages induced inflammation in the lung tissue of mice,and significantly affected the proportion of alveolar macrophages and neutrophils.qPCR results showed that the expression of inflammatory factors and IFN-I was elevated in alveolar macrophages treated with E.coli-derived outer membrane vesicles,and the WB results showed that the protein related to the cGAS-STING signalling pathway was activated.In WT and Stinggt/gt mice treated with E.coli-derived outer membrane vesicles or E.coli-derived outer membrane vesicles with DNA removed,H&E staining and immunofluorescence showed that E.coli-derived outer membrane vesicles were able to lead to inflammation in the lungs of the WT mice.However,WT or Sting gt/gt mice showed no significant inflammation in lung tissue after E.coli OMVs(pretreated with DNase).DNA removed,and H&E staining showed no inflammation in lung tissues of WT or Stinggt/gt mice treated with E.coli-derived outer membrane vesicles with DNA removed.H&E staining showed a decrease in lung inflammation,and immunofluorescence showed an increase in the proportion of neutrophils.The above results suggest that E.coli-derived outer membrane vesicles DNA may induce IFN-I to promote lung inflammation in septic mice by activating the cGAS-STING signalling pathway.Conclusion:E.coli-derived outer membrane vesicles can be transferred from the intestine to the lungs through the compromised intestinal barrier of septic mice by a mechanism that may be mediated by activation of the cGAS-STING signalling pathway in alveolar macrophages by the DNA of E.coliderived outer membrane vesicles.of E.coli origin could be transferred to the lungs through the damaged intestinal tract.The E.coli-derived membrane vesicles were used to stimulate alveolar macrophages,and the mechanism of inflammation induction was further explored by qPCR,WB lung tissue immunofluorescence.E.coli-derived membrane vesicles and DNA-removed E.coliderived membrane vesicles were given to Stinggt/gt mice and WT mice to further analyse the potential mechanism of E.coli-derived membrane vesicles in septic lung injury.更多还原