哮喘和ABPA血清蛋白质生物标志物的筛选

1.深圳大学生命与海洋科学学院,广东深圳518071;2.汕头大学医学院广东省感染病与分子免疫病理重点实验室,广东汕头515041;3.复旦大学附属中山医院呼吸内科,上海200032

分子病理;哮喘;变应性支气管肺曲霉菌病;生物标志物;血清蛋白质;蛋白质芯片;质谱多反应监测技术;酶联免疫吸附测定法

Screening out serum protein biomarkers from both groups of asthma and ABPA patients
YANG Lei1,WANG Yun1,2,JIN Meiling3,SHUAI Diquan1,CAI Hui3,YE Ling3,LI Shuiming1,and SHEN Bo1

1.College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518071, Guangdong Province, P. R. China;2.Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical School, Shantou 515041, Guangdong Province, P. R. China;3.Department of Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, P. R. China.

molecular pathology; asthma; allergic bronchopulmonary aspergillosis; biomarkers; plasma protein; protein assay; multiple reaction monitoring; enzyme linked immunosorbent assay (ELISA)

DOI: 10.3724/SP.J.1249.2022.05538

备注

哮喘和变应性支气管肺曲霉菌病(allergicbronchopulmonaryaspergillosis,ABPA)的临床表现相似,极易造成因漏诊、误诊而延误治疗.为提高这两种疾病诊断的准确性,运用蛋白芯片技术,分别检测哮喘患者、ABPA患者和正常对照受试者的血清蛋白质;经过生物信息软件分析,筛选出具有诊断价值的候选蛋白质生物标志物;利用多反应监测(multiplereactionmonitoring,MRM)质谱和酶联免疫吸附测定法(enzymelinkedimmunosorbentassay,ELISA),对候选靶标进行单一样本扩大数量和验证;并采用受试者工作特征(receiveroperatingcharacteristic,ROC)曲线,评估候选蛋白质生物标志物的诊断价值.研究发现:与正常对照组比较,哮喘组有7个差异表达蛋白质,ABPA组有15个差异表达蛋白质;与哮喘组比较,ABPA组有15个差异表达的蛋白质.MRM法的验证结果显示:与正常对照组比较,哮喘组的SERPINF2、C6和HABP2蛋白呈现显著性差异表达,ABPA组的SERPINF2和F10呈现显著性差异表达;ABPA组与哮喘组比较,CPN2和IGLL5呈现显著性差异表达.ELISA法验证结果表明:ABPA患者血清的CSF1蛋白质水平明显低于哮喘患者的,而TNFRSF10C蛋白质水平明显高于哮喘患者的.ROC曲线结果显示:与正常对照组比较,哮喘组差异蛋白质C6的ROC曲线下面积(areaundercurve,AUC)为0.914,敏感度为73.3%,特异性为99.9%,ABPA组F10的ROC曲线AUC为0.871,敏感度为80.0%,特异性为85.7%;与哮喘组比较,ABPA组中CPN2和IGLL5联合诊断的AUC为0.867,敏感度为86.7%,特异性为80.0%.由此可见,新筛选出的蛋白质生物标志物与哮喘或ABPA的发生发展密切相关,具有诊断与鉴别诊断的潜能.
Clinical manifestations of asthma and allergic bronchopulmonary aspergillosis (ABPA) are very similar, so that they are easy to be misdiagnosed. To improve the diagnostic accuracy of these two diseases, the protein array was firstly applied for measuring serum proteins from asthma and ABPA patients,compared with normal control subjects in this study. Protein biomarkers associated to the disease processes and with diagnostic value were analyzed by bioinformatics. The multiple reaction monitoring mass spectrometry (MRM-MS) and enzyme linked immunosorbent assay (ELISA) were then used to verify the candidate biomarkers with enlarged size of samples. The diagnostic value of candidate proteins was additionally evaluated with the receiver operating characteristic (ROC) curve. Via the comparison analysis, the disease related-candidates were identified, including 7 differentially expressed proteins (DEPs) between asthma and normal groups, 15 DEPs between ABPA and normal groups, and 15 DEPs between ABPA and asthma groups. Using MRM method, SERPINF2, C6 and HABP2 proteins were showed differential expression significantly between asthma and normal groups, SERPINF2 and F10 were represented differential expression significantly between ABPA and normal control groups, and CPN2 and IGLL5 were displayed differential expression significantly between ABPA and asthma groups. Furthermore, ELISA validation data were also showed that serum CSF1 protein levels in ABPA patients were significantly lower than those in asthma patients, while TNFRSF10C levels in ABPA patients were significantly higher than those in asthma patients. ROC curve analysis revealed that the AUC of C6 were 0.9 with the sensitivity of 73.3% and the specificity of 99.9% in comparison between asthma and normal groups. The AUC of F10 was 0.871, with the sensitivity of 80.0% and the specificity of 85.7% in comparison between ABPA and normal groups. And the AUC of the combined diagnosis of CPN2 and IGLL5 was 0.867, with more sensitivity of 86.7% and the specificity of 80.0% in comparison those between ABPA and asthma groups. In conclusion, novel protein biomarkers in this study were closely related to the occurrence and development of asthma or ABPA, and might have potentials for assistant diagnostic and differential diagnosis of those two diseases in clinical application.
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