结核分枝杆菌潜伏感染者外周血单个核细胞转录组学研究  

Transcriptome study on peripheral blood mononuclear cells of latent tuberculosis infection individuals

作　　者：尚雪恬 董静 黄麦玲[2] 孙琦[1] 贾红彦[1] 张蓝月 刘秋月[3] 姚明旭 王颖超 姬秀秀 杜博平 邢爱英[1] 潘丽萍[1] Shang Xuetian;Dong Jing;Huang Mailing;Sun Qi;Jia Hongyan;Zhang Lanyue;Liu Qiuyue;Yao Mingxu;Wang Yingchao;Ji Xiuxiu;Du Boping;Xing Aiying;Pan Liping(Beijing Key Laboratory for Drug Resistant Tuberculosis Research,Beijing Chest Hospital,Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China;Department of Tuberculosis,Beijing Chest Hospital,Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China;Department of Intensive Care Unit,Beijing Chest Hospital,Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China)

机构地区：[1]首都医科大学附属北京胸科医院耐药结核病研究北京市重点实验室/北京市结核病胸部肿瘤研究所,北京101149 [2]首都医科大学附属北京胸科医院结核科/北京市结核病胸部肿瘤研究所,北京101149 [3]首都医科大学附属北京胸科医院重症医学科/北京市结核病胸部肿瘤研究所,北京101149

出　　处：《中国防痨杂志》2024年第4期449-460,共12页Chinese Journal of Antituberculosis

基　　金：国家自然科学基金(82172279,82100011);北京市自然科学基金(L234055)。

摘　　要：目的:通过对比分析结核分枝杆菌潜伏感染(latent tuberculosis infection,LTBI)者和健康人的外周血单个核细胞(peripheral blood mononuclear cells,PBMC)经结核特异性抗原刺激后的转录组,寻找LTBI人群中结核抗原特异的免疫应答重要基因及可能发挥功能的信号通路,绘制LTBI基因表达谱。方法:应用微阵列芯片检测2009年12月在首都医科大学附属北京胸科医院招募的4例LTBI者(LTBI组)和4名健康人(健康对照组)经结核抗原刺激后的PBMC转录组,并对部分差异基因进行qPCR检测以验证芯片数据。应用加权基因共表达网络分析(weighted gene co-expression network analysis,WGCNA)筛选与LTBI最相关模块,并对模块中的基因分别进行基因本体论(geneontology,GO)分析、京都基因与基因组百科全书(Kyoto encylopedia of genes and genomes,KEGG)通路分析。通过基因集富集分析(gene set enrichment analysis,GSEA)从全转录组水平筛选信号通路。通过免疫浸润分析寻找与LTBI相关的免疫细胞。通过STRING数据库蛋白相互作用(protein-protein interaction,PPI)网络分析绘制两模块中差异基因相互作用网络图。结果:LTBI组与健康对照组间共发现393个差异表达基因(差异倍数>2或<0.5,P<0.05),LTBI中112个表达下调,281个表达上调。针对10个差异基因的实时荧光定量PCR(quantitative real-time PCR,qPCR)检测证实基因表达趋势与芯片数据一致,即CAMK1G、IL2、SPINK1、SUCNR1、HCAR3、IL18BP、CHI3L1、TNF均上调;RASGRP2、TPM2均下调。通过WGCNA分析确定与LTBI最相关的正相关蓝色模块(cor=0.88,P=0.004)和负相关蓝绿色模块(cor=―0.93,P=0.001)。对上述模块内基因进行KEGG富集分析,并行基于全转录组的GSEA分析,发现趋化因子信号通路(P<0.001)和凋亡信号通路(P=0.003)在两种分析中均明显富集。对两模块中的差异基因通过STRING数据库构建PPI网络图,通过内置插件计算得到每个模块中的前10个关键基因,即�Objective:The aim of the study is to explore the tuberculosis(TB)antigen specific transcriptome profile of latent TB infection(LTBI)individuals and identify the critical gene modules and pathways that may play significant roles in LTBI occurrence and development,by comparing the transcriptome results of peripheral blood mononuclear cells(PBMC)stimulated by TB-specific antigen of healthy controls(HC)and LTBI.Methods:Microarray test was used to uncover the transcriptome profile of PBMC stimulated by TB antigens in 4 cases of LTBI and 4 HC from Beijing Chest Hospital,Capital Medical University in December 2009,and further validated with qPCR to confirm the microarray data.Weighted gene co-expression network analysis(WGCNA)was used to identify the critical gene modules that were associated with LTBI.Geneontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were carried out based on the genes in the two modules significantly associated with LTBI.At the same time,GSEA-based KEGG analysis was also used in order to fully understand the transcriptome changes of PBMC after TB-specific antigen stimulation in the two groups.Immune infiltration analysis was also used to further confirm the important immune cells involving in LTBI occurrence.Protein-Protein Interaction(PPI)network analysis based on the STRING database was used to draw the differential gene interaction network diagram in the two modules.Results:A total of 393 differentially expressed genes were found between LTBI group and healthy group(fold change>2 or<0.5,P<0.05),of which 112 were down-regulated and 281 were up-regulated.qPCR analysis of 10 differential genes confirmed that the gene expression pattern was consistent with that of the microarray data,and CAMK1G,IL2,SPINK1,SUCNR1,HCAR3,IL18BP,CHI3L1 and TNF were up-regulated,while RASGRP2 and TPM2 were down-regulated.According to the analysis of WGCNA,the blue module with positive correlation(cor=0.88,P=0.004)and the turquoise module with negative correlation(cor=―0.93,P=0.001)

关 键 词：分枝杆菌 结核 分枝杆菌感染 转录因子 生物学标记 免疫 

分 类 号：R52[医药卫生—内科学]