[1]孙家官,瞿蓉蓉,孟伟民,等.结核分枝杆菌感染免疫调控关键基因筛选[J].新乡医学院学报,2023,40(11):1032-1038.[doi:10.7683/xxyxyxb.2023.11.005]
　SUN Jiaguan,QU Rongrong,MENG Weimin,et al.Screening the key immunoregulatory genes in Mycobacterium tuberculosis infection[J].Journal of Xinxiang Medical University,2023,40(11):1032-1038.[doi:10.7683/xxyxyxb.2023.11.005]
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结核分枝杆菌感染免疫调控关键基因筛选
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
40卷
期数:
2023年11
页码:
1032-1038
栏目:
临床研究
出版日期:
2023-11-05

文章信息/Info

Title:
Screening the key immunoregulatory genes in Mycobacterium tuberculosis infection
作者:
孙家官1; 瞿蓉蓉1; 孟伟民2; 崔俊伟3; 王志霞3; 宋杰1
(1.新乡医学院公共卫生学院，河南　新乡　453003；2.青海省第四人民医院重症医学科，青海　西宁　810000；3.新乡医学院第一附属医院结核内科，河南　卫辉　453100)
Author(s):
SUN Jiaguan1; QU Rongrong1; MENG Weimin2; CUI Junwei3; WANG Zhixia3; SONG Jie1
(1.School of Public Health,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;2.Department of Critical Care Medicine,the Fourth People′s Hospital of Qinghai Province,Xining 810000,Qinghai Province,China;3.Department of Tuberculosis Internal Medicine,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China)
关键词:
结核病; 差异表达基因; 生物信息分析; 免疫调控
Keywords:
tuberculosis; differentially expressed genes; bioinformatics analysis; immune regulation
分类号:
R392.11
DOI:
10.7683/xxyxyxb.2023.11.005
文献标志码:
A
摘要:
目的　筛选结核感染的免疫调控基因，为结核病诊断和治疗提供新靶标。
方法　选择2019年7月至9月青海省第四人民医院收治的5例肺结核患者为研究对象，分别于初诊（进展期）和抗结核治疗6个月后（好转期）用PAXgene全血RNA管采集外周血进行转录组测序，筛选差异表达基因，行基因本体（GO）分析、京都基因和基因组百科全书（KEGG）通路富集分析和免疫浸润分析。通过蛋白质互作数据库（STRING）构建蛋白-蛋白互作（PPI）网络，利用Cytoscape软件筛选结核病免疫调控的关键基因，在GSE83456数据集中进行关键基因诊断效能验证。
结果　共获得差异表达基因187个，其中上调基因149个，下调基因38个；差异表达基因主要富集于白细胞与细胞黏附及调节、细胞因子产生、T细胞激活的调节等生物过程，吞噬囊泡、溶细胞颗粒、T细胞受体复合物、次级颗粒、白明胶酶颗粒等细胞组分，以及主要组织相容性复合体（MHC）Ⅰb受体活性、二氧化碳转运复合体活性、MHCⅠ类蛋白复合物结合受体、MHCⅠ类受体活性、铵跨膜转运体活性等分子功能。KEGG富集分析显示，差异表达的基因主要涉及自然杀伤细胞介导的细胞毒性、辅助性T细胞（Th）1/Th2细胞分化、疟疾、Th17细胞分化、凋亡等信号通路。PPI网络分析发现，穿孔素-1（PRF-1）、白细胞分化抗原2（CD2）、白细胞分化抗原247（CD247）、杀伤细胞凝集素样受体D1（KLRD1）、杀伤细胞凝集素样受体B1（KLRB1）、自然杀伤细胞颗粒蛋白7（NKG7）、颗粒溶素（GNLY）、T-box转录因子21（TBX21）、白细胞介素2受体亚基β（IL2RB）、颗粒酶H（GZMH）是结核病免疫调控的关键基因。受试者操作特征曲线分析结果显示，KLRB1诊断肺结核的曲线下面积（AUC）为0.855 4（95%置信区间：0.783 2~0.927 6），敏感度为82.22%，特异度为75.41%；CD2诊断肺结核的AUC为0.835 7（95%置信区间：0.757 8~0.913 6），敏感度为62.22%，特异度为91.80%；CD247诊断肺结核的AUC为0.865 9（95%置信区间：0.795 9~0.935 9），敏感度为73.33%，特异度为86.89%。免疫细胞浸润的主成分分析结果显示，结核进展过程中免疫细胞浸润模式存在明显的群体异质性，进展期主要由嗜酸性粒细胞、中性粒细胞、巨噬细胞等固有免疫细胞发挥抗结核作用，好转期主要是由CD8+和CD4+T细胞、Th细胞等适应性免疫细胞以及激活的自然杀伤（NK）细胞发挥抗结核作用。免疫细胞浸润丰度分析显示，单核细胞和中性粒细胞在免疫微环境丰度最高；好转期记忆B细胞、嗜酸性粒细胞、M1型巨噬细胞的丰度显著降低，而幼稚B细胞、静息态CD4+记忆T细胞、静息态NK细胞、激活态NK细胞的丰度显著升高（P<0.05）。
结论　结核患者疾病进展过程中差异表达基因与免疫应答密切相关，细胞毒性相关基因PRF1、KLRD1、KLRB1、NKG7、GNLY、TBX21、GZMH和T细胞活化信号转导基因CD2、CD247、IL2RB是结核病免疫调控的关键基因，有望成为结核诊断和治疗的潜在靶标。
Abstract:
Objective　To screen immune regulatory genes,provide new targets for tuberculosis diagnosis and treatment.
Methods　Five pulmonary tuberculosis patients admitted to the Fourth People′s Hospital of Qinghai Province from July to September 2019 were selected as the research subjects.Peripheral blood of patients at initial diagnosis (progression stage) and 6 months after anti-tuberculosis treatment (improvement stage) was collected by using PAXgene whole blood RNA tubes for transcriptome sequencing.Differentially expressed genes were screened for Gene Ontology (GO) analysis,Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and immune infiltration analysis.A protein-protein interaction (PPI) network was constructed through the Search Tool for Recurring Instances of Neighbouring Genes database,key immunoregulatory genes was searched by subsequently Cytoscape software.The diagnostic efficacy of screened key genes was validated in the GSE83456 dataset.
Results　A total of 187 differentially expressed genes were obtained,including 149 up-regulated genes and 38 down-regulated genes;differentially expressed genes were mainly enriched in biological processes such as leukocyte cell adhesion and regulation,cytokine production,and regulation of T cell activation;cell components such as phagocytic vesicles,lysosomes,T cell receptor complexes,secondary particles,and white gelatin enzyme particles;as well as molecular functions such as the activity of major histocompatibility complex (MHC) Ib receptor,carbon dioxide transport complex,and MHC class I protein complex binding receptor,MHC class I receptor activity and ammonium transmembrane transporter.KEGG enrichment analysis showed that differentially expressed genes mainly involved signaling pathways such as cytotoxicity mediated by natural killer cells,helper T cell (Th) 1/Th2 cell differentiation,malaria,Th17 cell differentiation and apoptosis.PPI network analysis found that perforin-1(PRF-1),clusters of differentiation 2(CD2),clusters of differentiation 247(CD247),killer cell lectin like receptor D1(KLRD1),killer cell lectin like receptor B1(KLRB1),natural killer cell granule protein 7(NKG7),granulysin(GNLY),T-box transcription factor 21(TBX21),interleukin 2 receptor subunit beta(IL2RB) and granzyme H(GZMH) were the key genes for immune regulation of tuberculosis.The analysis of the operator characteristic curve showed that the area under the curve (AUC) of KLRB1 for diagnosing pulmonary tuberculosis was 0.855 4 (95% confidence interval:0.783 2-0.927 6),with a sensitivity of 82.22% and specificity of 75.41%;the AUC of CD2 for diagnosing pulmonary tuberculosis was 0.835 7 (95% confidence interval:0.757 8-0.913 6),with a sensitivity of 62.22% and specificity of 91.80%;the AUC of CD247 for diagnosing pulmonary tuberculosis was 0.865 9 (95% confidence interval:0.795 9-0.935 9),with a sensitivity of 73.33% and specificity of 86.89%.The principal component analysis results of immune cell infiltration showed that there was significant population heterogeneity in the infiltration pattern of immune cells during the progression of tuberculosis.During the progression stage,innate immune cells such as eosinophils,neutrophils and macrophages played an anti-tuberculosis role;while during the improvement stage,adaptive immune cells such as CD8+ and CD4+ T cells,Th cells,and activated natural killer (NK) cells played an anti-tuberculosis role.The abundance analysis of immune cell infiltration showed that monocytes and neutrophils had the highest abundance in the immune microenvironment.The abundance of memory B cells,eosinophils,and M1 macrophages significantly decreased during the improvement period;while the abundance of immature B cells,resting CD4+ memory T cells,resting NK cells,and activated NK cells significantly increased (P<0.05).
Conclusion　Differentially expressed genes in tuberculosis progression are involved in immune response.Cytotoxicity-related genes such as PRF1,KLRD1,KLRB1,NKG7,GNLY,TBX21,GZMH,and T-cell signal transduction related genes such as CD2,CD247 and IL2RB are key immunoregulatory genes in tuberculosis,and they are expected to be potential targets for the diagnosis and treatment of tuberculosis.

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更新日期/Last Update: 2023-11-05