摘要
目的在单细胞水平上分析锌稳态相关基因及相关细胞在炎症性肠病(IBD)患者肠黏膜中的表达,评估其在预测IBD患者抗肿瘤坏死因子(TNF)治疗反应的价值。方法收集4个基因表达综合(GEO)数据库中75例回肠或结直肠活检样本的单细胞RNA测序数据,包括克罗恩病(CD)、溃疡性结肠炎(UC)患者以及正常对照者(NC)。在R语言环境下,首先通过无监督聚类分析将IBD细胞分群,再采用锌稳态相关基因评分评估各细胞群的锌稳态情况,鉴定出与锌稳态相关基因密切相关的细胞群,即金属硫蛋白相关巨噬细胞(MT Mph)。随后收集中山大学附属第一医院收治的IBD患者及健康体检人群的结肠组织进行免疫荧光(IF)染色,验证比较IBD和NC组织中MT Mph数量差异。为进一步探讨MT Mph的功能和来源,在R语言环境下,比较GEO数据库MT Mph和非金属硫蛋白相关巨噬细胞(non-MT Mph)特征基因,通过京都基因与基因组百科全书(KEGG)富集分析进一步富集特征基因,采用细胞通讯分析MT Mph与不同细胞间的通讯机制,采用拟时序分析MT Mph来源及相关的信号通路,分析对比单核细胞、MT Mph与non-MT Mph之间的转录因子差异。通过单样本基因集富集分析(ssGSEA)评估MT Mph特征基因的表达,采用ssGSEA和抗TNF治疗反应构建模型,评价MT Mph特征基因对IBD患者抗TNF治疗反应的预测效能。结果IBD细胞聚类注释为7个主要细胞群,分别为T细胞、B细胞、浆细胞、髓系细胞、成纤维细胞、内皮细胞和上皮细胞。IBD髓系细胞锌稳态相关基因评分高于NC组。髓系细胞可分为单核细胞、巨噬细胞、中性粒细胞和树突状细胞。根据细胞表达锌稳态基因特别是高表达金属硫蛋白基因的情况,可进一步将巨噬细胞分为MT Mph和non-MT Mph,且IBD肠道MT Mph的数量较NC组显著增加。通过IF验证,每个高倍镜视野下UC和CD中CD68+MT1G+阳性细胞(MT Mph)数量显著多于NC组(UC比NC:30.80±7.29比9.80±1.80,P<0.001;CD比NC:36.00±9.30比9.80±1.80,P<0.001)。KEGG通路富集分析发现,MT Mph差异基因富集出炎症相关通路的关键基因,特别是TNF信号通路;细胞通讯分析发现MT Mph与其他细胞在IBD的TNF信号通路较NC显著增强。拟时序分析结果显示,单核细胞可分化为MT Mph,且分化过程中金属硫蛋白基因(MT2A、MT1X、MT1H和MT1G)的表达显著上调。转录因子分析表明,MT Mph转录因子SMARCB1以及ZMYND8活性较单核细胞显著升高,经典炎症转录因子HIF1A、STAT3、NFKB1活性较non-MT Mph显著升高。根据ssGSEA和TNF治疗反应分别构建CD和UC相应的预测模型[CD:曲线下面积(AUC)=0.966,P<0.01;AUC=0.793,P<0.01]。验证结果显示,该模型不能预测CD和UC患者对维得利珠单克隆抗体的治疗反应(均P>0.05)。结论IBD肠道存在锌稳态失衡,MT Mph是一组高度表达锌稳态相关基因的细胞群,其与TNF炎症通路密切相关。以MT Mph特征基因构建的预测模型可能可预测IBD抗TNF的治疗反应。
Objective:To analyze the expression of zinc homeostasis-related genes and related cells in the intestinal mucosa of inflammatory bowel disease(IBD)patients at the single-cell level and to evaluate their value in predicting the response to anti-tumor necrosis factor(TNF)therapy in IBD patients.Methods:Single-cell RNA sequencing data from 75 ileal or colorectal biopsy samples,including those from patients with Crohn's disease(CD),ulcerative colitis(UC),and normal controls(NC),were collected from four gene expression omnibus(GEO)databases.Unsupervised clustering analysis in R language was employed to classify IBD cells,zinc homeostasis-related gene scores were used to assess the zinc homeostasis status of different cell clusters,and the cell clusters closely related to zinc homeostasis-related genes,namely metallothionein-associated macrophages(MT Mph),were identified.Then the colon tissues from IBD patients and healthy individuals treated at the First Affiliated Hospital of Sun Yat-sen University were collected for immunofluorescence(IF)staining to compare the differences in MT Mph numbers between IBD and NC tissues.To further explore the function and origins of MT Mph,the characteristic genes of MT Mph and non-metallothionein-associated macrophages(non-MT Mph)from database were compared,the Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis was further used to enrich the characteristic genes,cell communication analysis was used to investigate the communication mechanisms between MT Mph and different cells,Quasi-time sequence was used to explore the origin of MT Mph and related signaling pathways,and the differences in transcription factors among monocytes,MT Mph and non-MT Mph were analyzed in R language.Single sample gene set enrichment analysis(ssGSEA)was used to evaluate the expression of MT Mph characteristic genes,and ssGSEA combined with the response to anti-TNF were used to construct the model in order to explore the predictive value of MT Mph characteristic genes for the response to anti-TNF therapy in IBD patients.Results:IBD cells were clustered and annotated into seven major cell clusters,namely T cells,B cells,plasma cells,myeloid cells,fibroblasts,endothelial cells,and epithelial cells.The results of zinc homeostasis-related gene scores showed that the scores of IBD myeloid cells were higher than those of the NC group.Myeloid cells could be divided into monocytes,macrophages,neutrophils,and dendritic cells.Based on the expression of zinc homeostasis genes,especially the high expression of metallothionein genes,the macrophages were divided into MT Mph and non-MT Mph,and the number of MT Mph in the IBD intestine was significantly increased compared to the NC group.IF validation showed that the number of CD68+MT1G+cells(MT Mph)in both UC and CD were significantly higher than that in the NC group under per high-power field of view(UC vs.NC:30.80±7.29 vs.9.80±1.80,P<0.001;CD vs.NC:36.00±9.30 vs.9.80±1.80,P<0.001).KEGG pathway enrichment analysis revealed that the differential genes of MT Mph were enriched in key genes of inflammation-related pathways,especially the TNF signaling pathway.Cell communication analysis showed that the TNF signaling pathway between MT Mph and other cells in IBD was significantly enhanced compared to NC.Quasi-time sequence analysis results showed that monocytes could differentiate into MT Mph,and the expression of metallothionein genes(MT2A,MT1X,MT1H and MT1G)was significantly upregulated during the differentiation process.Transcription factor analysis showed that the transcription factors SMARCB1 and ZMYND8 of MT Mph were significantly higher than those of monocytes,and the classical inflammatory transcription factors HIF1A,STAT3,and NFKB1 were significantly higher than those of non-MT Mph.Prediction models for CD and UC were constructed respectively based on ssGSEA and TNF treatment response[CD:area under the curve(AUC)=0.966,P<0.01;AUC=0.793,P<0.01].Validation results showed that the model could not predict the response of CD and UC patients to vedolizumab therapy(both P>0.05).Conclusions:There is a zinc homeostasis imbalance in IBD intestine,and MT Mph are a group of cells with high expression of zinc homeostasis-related genes,which are closely related to the TNF inflammatory pathway.The prediction model constructed based on the characteristic genes of MT Mph may be able to predict the response to anti-TNF therapy in IBD.
作者
周高适
张牡丹
陈旻湖
张盛洪
Zhou Gaoshi;Zhang Mudan;Chen Minhu;Zhang Shenghong(Department of Gastroenterology,the First Afiliated Hospital,Sun Yat-sen University,Guangzhou 510080,China)
出处
《中华炎性肠病杂志(中英文)》
2025年第3期199-210,共12页
Chinese Journal of Inflammatory Bowel Diseases
基金
广东省基础与应用基础研究基金(2024A1515013032)。
关键词
炎症性肠病
锌稳态
金属硫蛋白相关巨噬细胞
单细胞RNA测序
抗肿瘤坏死因子
治疗反应
Inflammatory bowel disease
Zinc homeostasis
Metallothionein-associated macrophage
Single-cell RNA sequencing
Anti-tumornecrosisfactor
Response
作者简介
通信作者:张盛洪,Email:zhshh3@mail.sysu.edu.cn。
