非酒精性脂肪性肝炎小鼠模型肝组织T淋巴细胞的特征分析  

Characteristics of T cells in the liver tissues of mice with nonalcoholic steatohepatitis

作　　者：冒婷 徐铭益 王佳轶 MAO Ting;XU Mingyi;WANG Jiayi(Department of Gastroenterology,Shanghai East Hospital,Tongji University,Shanghai 200120,China;Department of Gastroenterology,Shanghai General Hospital,Shanghai Jiao Tong University,Shanghai 200080,China)

机构地区：[1]同济大学附属东方医院消化内科,上海200120 [2]上海交通大学附属第一人民医院消化内科,上海200080

出　　处：《临床肝胆病杂志》2025年第3期461-468,共8页Journal of Clinical Hepatology

基　　金：上海市浦东新区卫生健康委员会医学学科建设项目(PWYgf2021-02)。

摘　　要：目的采用单细胞测序技术揭示非酒精性脂肪肝炎(NASH)小鼠肝组织T淋巴细胞单细胞水平的异质性和转录组学特征,为研究T淋巴细胞在NASH中的作用机制提供新的依据。方法6只C57BL/6雄性小鼠随机分为普通饲料喂养的对照组和胆碱蛋氨酸缺乏饲料喂养的NASH组,每组各3只。造模6周后取小鼠肝组织进行单细胞RNA测序。分析T淋巴细胞单细胞亚群的特异性差异表达基因,分别行降维聚类、细胞类型注释、t分布随机邻域嵌入(t-SNE)、小提琴图、基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。利用免疫荧光染色观察两组小鼠肝脏中Tcrα(T淋巴细胞分子标志)、Tcf7、Cxcr6(特征标志基因)的表达情况。计量资料两组间比较采用成组t检验。结果小鼠肝脏中共鉴定出2个T淋巴细胞亚群:(1)第6簇T淋巴细胞占比从对照组的58.5%降低到NASH组的48.7%。前4位特异性基因包括Nsg2、Cd8b1、Cd8a和Tcf7。该簇65%的细胞表达Tcf7(第6簇特征标志基因),定义为Tcf7+T淋巴细胞亚群。GO和KEGG富集分析显示,它们参与调控T淋巴细胞活化、白细胞黏附、结合泛素样蛋白连接酶,以及辅助性T淋巴细胞(Th)17、Th1、Th2细胞分化等信号通路。(2)第7簇T淋巴细胞占比从对照组的41.5%增高到NASH组的51.3%。前4位特异性基因包括Cd40lg、Tcrg-C1、Il2rα和Cxcr6。该簇90%的细胞表达Cxcr6,定义为Cxcr6+T淋巴细胞亚群。GO和KEGG富集分析提示,该亚群参与调控T淋巴细胞活化、细胞因子产生,以及T淋巴细胞受体信号通路、Th17细胞分化与MAPK信号通路。免疫荧光结果显示,与对照组相比,NASH组小鼠肝脏中Tcf7蛋白和Tcrα蛋白共染阳性区域减少(1.80%±0.67%vs 0.33%±0.13%,P<0.05),而Cxcr6蛋白和Tcrα蛋白共染阳性区域增加(0.50%±0.09%vs 2.66%±0.33%,P<0.001)。结论NASH小鼠肝脏中Tcf7+T淋巴细胞的占比降低,而Cxcr6+T淋巴细胞的占比增高,揭示�Objective To investigate the heterogeneity and transcriptomic characteristics of T-cell subsets in the liver of mice with nonalcoholic steatohepatitis(NASH)at the single-cell level using single-cell RNA sequencing(scRNA-seq),and to provide a reference for studying the mechanism of action of T cells in NASH.Methods Six male C57BL/6 mice were randomly divided into control group fed with regular diet and NASH group fed with methionine-choline-deficient(MCD)diet,with three mice in each group,and liver tissue was collected for scRNA-seq after 6 weeks of modeling.Specific differentially expressed genes were analyzed between T-cell subsets,and related analyses were performed,including dimension clustering,cell type annotation,tdistributed stochastic neighbor embedding(t-SNE),violin plot,gene ontology(GO)functional enrichment analysis,and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Immunofluorescent staining was used to observe the expression of the T cell marker Tcrαand the specific marker genes Tcf7 and Cxcr6 in the liver of mice in the two groups.The independent-samples t test was used for comparison of continuous data between two groups.Results Two T cell subsets were identified in the liver of mice,and the percentage of cluster 6 decreased from 58.5%in the control group to 48.7%in the NASH group.The top four specific genes were Nsg2,Cd8b1,Cd8a,and Tcf7.Tcf7,a characteristic marker gene for cluster 6,was expressed in 65%of cells in cluster 6,and therefore,cluster 6 was defined as Tcf7+T cells.The GO and KEGG enrichment analyses showed that the differentially expressed genes of cluster 6 were involved in T cell activation,leukocyte adhesion,binding ubiquitin-like protein ligase,and the signaling pathways for Th17,Th1,and Th2 cell differentiation.The percentage of cluster 7 increased from 41.5%in the control group to 51.3%in the NASH group.The top four specific genes of cluster 7 were Cd40lg,Tcrg-C1,Il2rα,and Cxcr6.Cxcr6 was expressed in 90%of cells in cluster 7,and therefore,cluster 7 wa

关 键 词：非酒精性脂肪性肝炎 T淋巴细胞 单细胞测序 小鼠 近交C57BL 

分 类 号：R73[医药卫生—肿瘤]