【作者】 林平；

【导师】 梁宗安；

【作者基本信息】 四川大学 ， 内科学（呼吸病学）（专业学位）， 2023， 博士

【摘要】 研究目的:哮喘是临床常见的气道慢性炎症性疾病之一,其典型的临床症状为反复发作的喘息、胸闷、气促或咳嗽。近半个世纪以来,哮喘发病率在世界范围内持续增加,给家庭及社会造成的负担与日俱增。哮喘发病机制至今尚未完全阐明。临床常常使用吸入性糖皮质激素控制气道炎症及症状,但糖皮质激素无法从根本上解决患者气道炎症和免疫功能紊乱,且部分患者即使长期规律使用糖皮质激素也不能有效控制其哮喘症状。故进一步明确哮喘气道炎症的发生机制,寻找抗炎治疗的新靶点是哮喘研究亟需解决的问题。哮喘气道炎症由多种细胞共同调控,因此潜在的细胞间信息沟通是关键。外泌体最早发现于1986年,是一种直径约30-150纳米的圆形单层膜结构的小囊泡,能被机体内大多数细胞分泌。外泌体携带了许多生物活性物质,是细胞间通讯的重要介质,参与了包括哮喘在内的多种呼吸道疾病的发生。现有的研究发现嗜酸性粒细胞、支气管上皮细胞、淋巴细胞等细胞来源外泌体可以促进哮喘气道炎症的形成。然而,少有研究关注血管内皮细胞外泌体对哮喘气道炎症的影响。明确血管内皮细胞外泌体在哮喘中的作用将有助于阐明哮喘气道炎症的发生机制及寻找抗炎治疗新靶点。我们前期利用生物信息分析发现在哮喘血清外泌体中,miR-126-5p含量显著升高。miR-126-5p是miR-126的成熟体,特异性高表达于血管内皮细胞。我们推测血管内皮细胞在过敏源的刺激下可以释放含miR-126-5p的外泌体,从而调控哮喘气道炎症。因此,本研究从体内及体外实验两部分展开研究。通过建立哮喘小鼠模型及屋尘螨（house dust mite,HDM）刺激人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）作为研究对象,探索血管内皮细胞在过敏源的刺激下是否分泌含miR-126-5p的外泌体调控哮喘气道炎症及潜在机制。材料和方法:1.生物信息分析:从公共数据库（Gene Expression Omnibus）下载小鼠血清外泌体中非编码RNA高通量测序数据（GSE121818,Illumina Hi Seq 2500）,分析哮喘组和对照组两组间小鼠血清外泌体中miRNAs的表达情况。2.利用卵清蛋白（OVA）和氢氧化铝[AL（OL）₃]构建哮喘小鼠模型。提取小鼠血清外泌体,通过RT-PCR验证miRNA-126-5p在外泌体中的表达情况。体外培养HUVECs,使用si NC和simiR-126-5p慢病毒转染HUVECs,从而获得富含miR-126-5p的细胞外泌体及其对照。提取细胞外泌体并气道过继给哮喘模型小鼠,通过肺组织病理染色、小鼠肺功能和炎症因子表达评估额外增加含有miR-126-5p的外泌体对哮喘小鼠表型的影响。此外,我们也利用miR-126-flox小鼠敲除miR-126基因,以降低血管内皮细胞及外泌体中miR-126-5p表达。通过肺组织切片病理染色、小鼠肺功能和炎症因子评估降低miR-126-5p对哮喘小鼠表型的影响。3.对miR-126敲除小鼠的肺组织送m RNA测序并进行生物信息学分析,找到了miRNA-126-5p调节哮喘气道炎症的潜在靶基因TET2。我们通过双荧光素酶实验、RT-PCR和WB验证了miRNA-126-5p对靶基因TET2的调节作用。为了探究血管内皮细胞是否通过外泌体miRNA-126-5p调控巨噬细胞内TET2基因的表达,我们在transwell室中共培养HUVECs和巨噬细胞并使用GW4869来干扰HUVECs外泌体的生成。通过检测巨噬细胞内miRNA-126-5p的含量和TET2的表达来评估外泌体miRNA-126-5p对巨噬细胞TET2表达的影响。4.最后利用流式细胞术及免疫组化染色探讨了miRNA-126-5p对血管内皮细胞与巨噬细胞间黏附的影响,以及对哮喘小鼠肺内巨噬细胞数量的影响。结果:1.生物信息分析发现miRNA-126-5p在哮喘模型小鼠血清外泌体中升高。2.成功建立哮喘小鼠模型。与对照组相比,哮喘模型组小鼠气道炎症加重,气道粘性分泌物增多,气道阻力增大和炎症因子（IL-1β、IL-4、IL-5）表达增多。3.成功分离小鼠血清外泌体,外泌体在电镜下形态为椭圆形。miRNA-126-5p在哮喘模型组小鼠血清外泌体中含量增加。4.成功将si NC和simiR-126-5p慢病毒转染HUVECs。和simiR-126-5p组相比,si NC组HUVECs和细胞上清液外泌体中miR-126-5p含量显著升高。提取细胞外泌体并气道过继给哮喘模型小鼠。和simiR-126-5p外泌体过继组小鼠相比,si NC组小鼠伴随加重的气道周围炎症,增加的气道粘性分泌物,升高的气道阻力和细胞因子表达（IL-1β、IL-4）。5.利用miR-126-flox小鼠成功敲除miR-126基因。miR-126敲除小鼠血管内皮细胞及血清外泌体中miRNA-126-5p表达降低。miR-126敲除哮喘小鼠气道炎症减轻,气道阻力降低和IL-4表达降低。6.哮喘模型小鼠miR-126基因敲除后,KEGG显示差异表达基因主要涉及细胞因子-细胞因子受体通路。生物信息分析示TET2可能是miRNA-126-5p发挥作用的靶基因。7.miR-126-5p可以特异性与靶基因TET2 3’端非编码区结合并调控巨噬细胞内TET2的表达。8.HDM刺激可诱导miR-126-5p在HVUECs外泌体的中表达。HVUECs外泌体miR-126-5p可被巨噬细胞吸收并降低巨噬细胞内TET2的表达。9.miRNA-126-5p不会影响血管内皮细胞-巨噬细胞间黏附且其不能改变哮喘模型小鼠肺内巨噬细胞数量。结论:1.血管内皮细胞特异性miR-126-5p在哮喘小鼠血清外泌体中含量升高。外泌体miR-126-5p可以促进OVA诱发的哮喘气道炎症。2.外泌体miR-126-5p可能通过调节TET2的表达从而影响哮喘的气道炎症。3.外泌体miR-126-5p可调节巨噬细胞TET2的表达,但对哮喘小鼠肺内巨噬细胞数量无明显影响。更多还原

【Abstract】 Background and Objective:Asthma is one of the common chronic inflammatory diseases of the airways in the clinic,and its typical clinical symptoms are recurrent wheezing,chest tightness,shortness of breath,or cough.For nearly half a century,the incidence of asthma has continued to increase worldwide,and the burden on families and society is increasing daily.The pathogenesis of asthma has not yet been fully elucidated.Inhaled glucocorticoids are often used clinically to control airway inflammation,but glucocorticoids cannot fundamentally resolve airway inflammation and immune dysfunction in patients,and some patients cannot effectively control their asthma symptoms even with long-term regular use of glucocorticoids.Therefore,further clarification of the pathogenesis of airway inflammation in asthma and finding new targets for anti-inflammatory therapy are problems that need to be solved urgently.Asthmatic airway inflammation is jointly regulated by various cells in the lung,so the potential intercellular information communication is the key.Exosomes were first discovered in 1986.They are small vesicles with a diameter of about 30-150 nm and a single-layer membrane structure,which can be secreted by most cell types.Exosomes carry many biologically active substances and are important mediators of intercellular communication.Exosomes participated in the occurrence of various respiratory diseases including asthma.Current studies had found that exosomes derived from eosinophils,bronchial epithelial cells,and lymphocytes could promote the formation of asthma.However,little research had focused on the role of vascular endothelial cell-derived exosomes in asthma.Clarifying the role of vascular endothelial cell-derived exosomes would help improve the airway inflammation mechanism of asthma and found new anti-inflammatory therapeutic targets.We previously used bioinformatic analysis to find that in asthmatic serum exosomes,the content of vascular endothelial-specific miR-126-5p was significantly increased.miR-126-5p is the mature form of miR-126,which is highly specifically expressed in vascular endothelial cells,but its role in asthma is still unclear.We speculated that vascular endothelial cells could release exosomes containing miR-126-5p under the stimulation of allergens,thereby regulating airway inflammation in asthma.Therefore,this study was carried out in two parts:in vivo and in vitro experiments.Through the establishment of the asthma mouse model and the stimulation of HUVECs by house dust mite（HDM）,we explored whether vascular endothelial cells participated in the regulation of asthma airway inflammation by secreting exosomal miRNA-126-5p and the potential mechanism.Materials and Methods:1.We downloaded the high-throughput sequencing data of non-coding RNA in mouse serum exosomes from the public database（Gene Expression Omnibus）（GSE121818,Illumina Hi Seq 2500）,and analyzed the expression of miRNAs in mouse serum exosomes between the asthma group and the control group.2.The asthma mouse model was constructed with ovalbumin（OVA）and aluminum hydroxide[AL（OL）₃].Mouse serum exosomes were extracted,and the expression of miRNA-126-5p in exosomes was detected by RT-PCR.HUVECs were cultured in vitro and transfected with si NC and simiR-126-5p lentivirus.Cellular exosomes were extracted and adopted into the airway of asthma model mice.The effects of additional exosomes containing miR-126-5p on the phenotype of asthmatic mice were evaluated by lung histopathological staining,mouse lung function,and inflammatory factors.In addition,we also used miR-126-flox mice to knock out the miR-126 gene to reduce the expression of miR-126-5p.The effects of reducing miR-126-5p on the phenotype of asthmatic mice were also evaluated by pathological staining of lung tissue sections,mouse lung function and inflammatory factors.3.The lung tissue of miR-126 knockout mice were sent for m RNA sequencing.We found that TET2 was a potential target gene of miRNA-126-5p regulating airway inflammation in asthma.We verified the regulatory effect of miRNA-126-5p on the target gene TET2 by dual luciferase assay,RT-PCR and WB.To explored whether vascular endothelial cells regulated the expression of TET2 in macrophages by secreting exosomes containing miRNA-126-5p,we co-cultured HUVECs and macrophages in a transwell chamber and GW4869 was used to reduce the production of exosomes.The effect of exosomal miRNA-126-5p on macrophage was evaluated by detecting the content of miRNA-126-5p and the expression of TET2 in macrophage.4.Finally,the effect of miRNA-126-5p on the adhesion between vascular endothelial cells and macrophages and the number of macrophages in the lungs of asthmatic mice were explored by flow cytometry and immunohistochemistry.Results:1.Bioinformatics analysis found that miRNA-126-5p was elevated in serum exosomes of the asthmatic model mouse.2.We successfully established an asthma mouse model.Compared with the control group,the asthma model group mouse showed that the inflammation around the airway was aggravated,the airway mucus secretion and airway resistance were increased,and the expression of cytokines（IL-1β,IL-4,IL-5）was also elevated significantly.3.We successfully isolated mouse serum exosomes,which were oval under the electron microscope.miRNA-126-5p was increased in the serum exosomes of asthmatic mice.4.HUVECs were transfected with si NC and simiR-126-5p lentivirus.The content of miR-126-5p in HUVECs and exosomes of the si NC group was significantly higher than that of the simiR-126-5p group.Exosomes were extracted and administered into the airway of the asthma model mouse.Compared with the simiR-126-5p group,the si NC group mice were associate with increased airway inflammation and airway viscous secretions,raised airway resistance,and higher expression of cytokines（IL-1βand IL-4）。5.The miR-126 gene was successfully knocked out using miR-126-flox mouse.The expression of miRNA-126-5p in vascular endothelial cells and serum exosomes of miR-126 knockout mice was reduced.miR-126 knockout mouse showed reduced airway inflammation,decreased airway resistance,and descending IL-4 level.6.After knocking out miR-126 in asthmatic mouse models,KEGG showed that differentially expressed genes were mainly involved in the cytokine-cytokine receptor pathway.Bioinformatics analysis suggested that the target gene of miRNA-126-5p might be TET2.7.miR-126-5p could specifically bind to the 3’non coding region of the target gene TET2 and reduce the expression of TET2 in macrophages.8.HDM could induce the expression of miR-126-5p in the exosomes of HVUECs.The exosomal miR-126-5p could be absorbed by macrophages and reduced the expression of TET2 in macrophages.9.miRNA-126-5p did not affect the adhesion between vascular endothelial cells and macrophages,and it could not alter the number of macrophages in the lungs of asthmatic mice.Conclusion:1.The content of endothelial-specific miR-126-5p increased in the serum exosomes of asthmatic mice.Exosomal miR-126-5p could exacerbate OVA-induced airway inflammation in asthmatic mice2.Exosomal miR-126-5p might affect airway inflammation in asthmatic mice by regulating the expression of TET2.3.Exosomes miR-126-5p could regulate the expression of TET2 in macrophage,but it could not alter the number of macrophages in the lungs of asthmatic mice.更多还原