【作者】 王静；

【导师】 董亮；

【作者基本信息】 山东大学 ， 内科学（呼吸系病）（专业学位）， 2023， 硕士

【摘要】 研究背景支气管哮喘(bronchial asthma,简称哮喘)是一种在发病年龄、相关危险因素、严重程度、合并症和治疗反应等方面具有异质性的慢性气道疾病。气道慢性炎症是哮喘发病的本质,是导致气道高反应性的重要机制,引起多变的可逆性气流受限。然而,随着病程的延长,气道长期持续性的炎症反复发作与修复,导致组织增生而发生气道重塑,形成不可逆的气流受限,致使哮喘具有难治性,增加了全球疾病负担。目前尚无治疗气道重塑的方法,早期控制气道炎症是阻止气道重塑发生的有效之策,除了糖皮质激素外,应用靶向炎症因子的生物制剂在治疗中重度哮喘中颇有成效。奥马珠单抗(Omalizumab)是首个应用于治疗中至重度过敏性哮喘的生物制剂,它是一种抗IgE单克隆抗体,主要通过降低游离IgE的水平控制气道炎症,但并非所有的患者在应用抗IgE单抗治疗后病情都可得到改善或控制,可能与哮喘复杂的机制和异质性有关。寻找可以预测哮喘患者对抗IgE单抗治疗反应的生物标志物可能有助于识别真正的临床获益人群,从而优化治疗、减轻疾病负担。研究目的通过简单易行的方法寻找潜在的可以预测抗IgE单抗临床疗效的生物标志物,以更好地指导哮喘患者应用抗IgE单抗,推动精准医疗的发展。研究方法在第一部分研究中,通过生物信息学的方法分析表达谱数据集GSE134544中对抗IgE单抗治疗应答和无应答的哮喘患者之间的差异表达基因(differentially expressed genes,DEGs),对DEGs进行GO富集分析和KEGG通路富集分析探究其潜在的生物学功能。通过构建PPI网络和功能模块筛选出两组患者DEGs中的核心基因,并绘制核心基因的箱线图与ROC曲线以检验其预测抗IgE单抗临床疗效的能力。通过免疫细胞浸润分析,探究核心基因在应答组与无应答组间存在显著差异的潜在机制。最后,利用Logistic回归分析构建核心基因的预测模型。在第二部分研究中,收集于我院接受抗IgE单抗治疗的哮喘患者的临床资料和开始治疗前的血清标本,依据疗程、疗效进行分组,采用酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测患者血清中核心基因表达产物的水平,通过ROC曲线分析其预测抗IgE单抗临床疗效的能力。研究结果在第一部分研究中,共鉴定出131个DEGs,这些基因主要与细胞因子及其受体的相互作用、细胞间通讯等功能有关,其中HBD、HBA1、HBE1、SELP、CXCL5、CXCR3、CD248、SPARC、FSTL1等9个核心基因在蛋白质水平存在明显且复杂的相互作用。在转录水平,无应答组中HBD、HBA1、HBE1、SELP、CXCL5、SPARC、FSTL1的表达均高于应答组,而CXCR3和CD248的表达均低于应答组,ROC曲线显示它们具有一定的预测效能。免疫细胞浸润分析显示,FSTL1与嗜酸性粒细胞、SELP与嗜酸性粒细胞、CXCL5与Th1细胞和Th2细胞之间存在显著的相关性。利用Logistic回归分析构建核心基因的预测模型发现,将CXCL5、CXCR3和HBA1共同纳入后,模型区分对抗IgE单抗治疗应答者和无应答者的能力更高。在第二部分研究中,检测了患者血清中FSTL1、SPARC、CXCL5和SELP的含量,分别探讨了哮喘患者抗IgE治疗疗程超过和未超过16周时的两种情况。根据哮喘总体控制水平将疗程超过16周的24名患者分为应答组(17人)和无应答组(7人),两组患者的基线人口学及临床特征无显著性差异,应答组患者治疗前后ACT评分的差值显著高于无应答组(P=0.005)。无应答组患者血清中FSTL1(P=0.003)、CXCL5(P=0.02)的含量显著高于应答组,ROC曲线显示曲线下面积(area under curve,AUC)分别是0.887、0.807,而两组患者血清中SPARC、SELP含量的差异无统计学意义。根据患者自行停止治疗的原因将疗程未超过16周的16名患者分为显效组(10人)和无效组(6人),无效组患者的平均年龄高于显效组(P=0.01),嗜酸性粒细胞计数的平均值低于显效组(P=0.012),其他人口学及临床特征无显著性差异。无效组患者血清中FSTL1(P=0.006)、CXCL5(P=0.03)的含量高于显效组,ROC曲线显示AUC分别是1.000、0.833,而两组患者血清中SPARC、SELP的含量也无统计学差异。研究结论在转录水平,基线时期外周血中CXCL5、HBA1的高表达和CXCR3的低表达可能与哮喘患者对抗IgE单抗的治疗无应答相关;在蛋白质水平,基线时期血清中的FSTL1和CXCL5可能可以作为生物标志物预测哮喘患者接受抗IgE单抗治疗的临床疗效。更多还原

【Abstract】 BackgroundBronchial asthma is a chronic airway disease that is heterogeneous in terms of age of onset,associated risk factors,severity,comorbidities,and response to treatment.Chronic inflammation of the airways is the essence of asthma pathogenesis and is an important mechanism leading to airway hyperresponsiveness,causing variable and reversible airflow limitation.However,with the prolongation of the disease,recurrent episodes of long-term persistent inflammation and repair of the airways lead to tissue proliferation and airway remodeling,resulting in irreversible airflow limitation,making asthma intractable and increasing the global disease burden.Currently,there is no treatment for airway remodeling.Early control of airway inflammation is an effective way to prevent airway remodeling.In addition to glucocorticoids,biologics targeting inflammatory factors have been effective in treating moderate to severe asthma.Omalizumab is the first biologic agent used in the treatment of moderate to severe allergic asthma.It is an anti-IgE monoclonal antibody,which mainly controls airway inflammation by reducing the level of free IgE.However,not all patients have improved or controlled with anti-IgE monoclonal antibody treatment,which may be related to the complex mechanism and heterogeneity of asthma.The search for biomarkers that predict response to anti-IgE monoclonal antibody therapy in asthma patients may help to identify populations of real clinical benefit,thereby optimizing treatment and reducing the disease burden.ObjectiveTo find potential biomarkers that can predict the clinical efficacy of anti-IgE monoclonal antibody through simple and feasible methods,so as to better guide the application of anti-IgE monoclonal antibody in asthma patients and promote the development of precision medicine.MethodsIn the first part of the study,differentially expressed genes(DEGs)between asthma patients responding to anti-IgE monoclonal antibody and those not responding to anti-IgE monoclonal antibody in the expression profiling dataset GSE134544 were analyzed by bioinformatics.The GO enrichment analysis and the KEGG pathway enrichment analysis were performed on DEGs to explore their potential biological functions.The core genes in DEGs of the two groups were screened by constructing a PPI network and functional modules,and the boxplot and ROC curve of the core genes were drawn to test their ability to predict the clinical efficacy of anti-IgE monoclonal antibody.Immunoinfiltration analysis was used to investigate the potential mechanism for the significant difference in core genes between the responder and non-responder groups.Finally,logistic regression analysis was used to construct a predictive model for the core gene.In the second part of the study,the clinical data of asthma patients treated with anti-IgE monoclonal antibody in our hospital and their serum sample before starting the treatment were collected.Patients were grouped according to treatment course and curative effect.The level of core gene expression products in the serum of patients was detected by enzyme-linked immunosorbent assay(ELISA),and its ability to predict the clinical efficacy of anti-IgE monoclonal antibody was analyzed by ROC curve.ResultsIn the first part of the study,131 DEGs were identified,which were mainly related to the interaction of cytokines and their receptors,cellular communication and other functions.Nine core genes,including HBD,HBAI,HBE1,SELP,CXCL5,CXCR3,CD248,SPARC and FSTL1,had significant and complex interactions at the protein level.At the transcription level,HBD,HBA1,HBE1,SELP,CXCL5,SPARC and FSTL1 in the non-responder group were higher than those in the responder group,while CXCR3 and CD248 in the non-responder group were lower than those in the responder group.The ROC curves show that core genes have some predictive efficacy.Immune cell infiltration analysis revealed significant correlations between FSTL1 and eosinophils,SELP and eosinophils,and CXCL5 and Th1 and Th2 cells.By constructing a predictive model,it was found that the model had a higher ability to distinguish between responders and non-responders to anti-IgE monoclonal antibody treatment when CXCL5,CXCR3,and HBA1 were included together.In the second part of the study,FSTL 1,SPARC,CXCL 5,and SELP were measured in patient serum.Two conditions were explored in asthma when anti-IgE treatment was over or not over 16 weeks.According to the overall level of asthma control,24 patients who were treated for more than 16 weeks were divided into a responder group(17 patients)and a nonresponder group(7 patients).There were no significant differences in baseline demographic and clinical characteristics between the two groups,and the changes in ACT scores before and after treatment were statistically different(P=0.005).The serum levels of FSTL1(P=0.003)and CXCL5(P=0.02)were significantly higher in the non-responder group than in the responder group,with ROC curves showing the area under the curve(AUC)of 0.887 and 0.807,respectively.The differences in serum levels of SPARC and SELP between the two groups were not statistically significant.The 16 patients who had not been treated for more than 16 weeks were divided into an effective group(10 patients)and an ineffective group(6 patients)according to their reasons for stopping treatment on their own.The mean age of the patients in the ineffective group was higher than that of the effective group(P=0.01),the mean eosinophil count was lower than that of the effective group(P=0.012),and there were no significant differences in other demographic and clinical characteristics.The serum levels of FSTL1(P=0.006)and CXCL5(P=0.03)were higher in the ineffective group than in the effective group,and the ROC curves showed AUC of 1.000 and 0.833,respectively.There was no statistical difference in serum SPARC and SELP between the two groups.ConclusionsAt the transcription level,high expression of CXCL5,HBA1 and low expression of CXCR3 in peripheral blood may be related to the non-response to anti-IgE monoclonal antibody therapy in asthmatic patients.At the protein level,serum levels of FSTL1 and CXCL5 at baseline may serve as biomarkers to predict the clinical efficacy of anti-IgE monoclonal antibody therapy in asthmatic patients.更多还原