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

冒婷; 徐铭益; 王佳轶

doi:10.12449/JCH250311

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

DOI: 10.12449/JCH250311

冒婷¹,
徐铭益¹,
王佳轶^2, , ,

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

基金项目:

上海市浦东新区卫生健康委员会医学学科建设项目 (PWYgf2021-02)

伦理学声明： 本研究方案于2022年3月1日经由上海市东方医院（同济大学附属东方医院）医学伦理委员会审批，批号：［2022］研预审第（054）号，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：冒婷负责实验和数据整理分析，撰写论文；徐铭益负责数据分析和撰写论文；王佳轶负责论文修改，对研究的思路和设计有关键贡献。

详细信息

通信作者:
王佳轶， 18017028830@163.com （ORCID： 0009-0004-8097-5288）

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出版历程
- 收稿日期: 2024-07-24
- 录用日期: 2024-09-23
- 出版日期: 2025-03-25

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

Ting MAO¹,
Mingyi XU¹,
Jiayi WANG^{2
, ,
,}

1.
Department of Gastroenterology，Shanghai East Hospital，Tongji University，Shanghai 200120，China
2.
Department of Gastroenterology，Shanghai General Hospital，Shanghai Jiao Tong University，Shanghai 200080，China

Research funding:

Medical Discipline Construction Project of Pudong Health Committee of Shanghai (PWYgf2021-02)

More Information

Corresponding author: WANG Jiayi， 18017028830@163.com （ORCID： 0009-0004-8097-5288）

摘要

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，揭示了NASH小鼠肝组织T淋巴细胞的特征和差异。
- 非酒精性脂肪性肝炎 /
- T淋巴细胞 /
- 单细胞测序 /
- 小鼠，近交C57BL
Abstract: 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， t-distributed 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 Tcrα and 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 was defined as Cxcr6⁺ T cells. The GO and KEGG enrichment analyses showed that cluster 7 was involved in T cell activation， cytokine production， the T cell receptor signaling pathway， and the Th17 cell differentiation and MAPK signaling pathway. Immunofluorescence assay showed that compared with the control group， the NASH group showed a significant reduction in the area with positive co-expression of Tcf7 protein and Tcrα protein （1.80%±0.67% vs 0.33%±0.13%， P<0.05） and a significant increase in the area with positive co-expression of Cxcr6 protein and Tcrα protein （0.50%±0.09% vs 2.66%± 0.33%， P<0.001）. Conclusion There is a reduction in the percentage of Tcf7⁺ T cells and an increase in the percentage of Cxcr6⁺ T cells in NASH mice， revealing the characteristics and differences of T cells in the liver of NASH mice.
- Non-Alcoholic Steatohepatitis /
- T-Lymphocytes /
- Single-cell RNA Sequencing /
- Mice， Inbred C57BL

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注： a，小鼠肝组织中T淋巴细胞亚群的t-SNE分析，其中左图显示21个细胞簇（绿色的2簇为T淋巴细胞簇），右图显示第6、7簇T淋巴细胞簇；b，第6、7簇T淋巴细胞在对照组和NASH组中的占比；c，热图显示第6、7簇T淋巴细胞前10位特异性基因。

图 1 NASH组小鼠肝组织T淋巴细胞亚群的分群和各亚群的变化

Figure 1. Definition of T cell subsets and changes in each subpopulation in the liver tissues of NASH mice

下载: 全尺寸图片幻灯片

注： t-SNE图（a）和小提琴图（b）显示第6簇T淋巴细胞的前4位特异性基因；第6簇T淋巴细胞的特异性基因的KEGG通路富集分析（c）和GO富集分析（d）。

图 2 第6簇T淋巴细胞的转录组学特征

Figure 2. The transcriptomic characteristics of Cluster 6

下载: 全尺寸图片幻灯片

注： t-SNE图（a）和小提琴图（b）显示第7簇T淋巴细胞的前4位特异性基因；第7簇T淋巴细胞的特异性基因的GO富集分析（c）和KEGG通路富集分析（d）。

图 3 第7簇T细胞的转录组学特征

Figure 3. The transcriptomic characteristics of Cluster 7

下载: 全尺寸图片幻灯片

注： DAPI蓝色荧光代表细胞核，Tcrα蛋白红色荧光代表T淋巴细胞。a，绿色荧光为Tcf7，黄色箭头指Tcf7⁺Tcrα⁺T淋巴细胞（×630）；b，Tcf7⁺Tcrα⁺T淋巴细胞占比；c，绿色荧光为Cxcr6，黄色箭头指Cxcr6⁺Tcrα⁺T淋巴细胞（×630）；d，Cxcr6⁺Tcrα⁺T淋巴细胞占比。

图 4 免疫荧光显示T淋巴细胞亚群及特征标志基因的变化

Figure 4. Immunofluorescence shows the changes of T cell subsets and of the expression of the most specific genes between the control and NASH group

下载: 全尺寸图片幻灯片

表 1 第6簇T细胞前10位特异性基因

Table 1. Top10 specific DEGs of Cluster 6

基因	P值	avg_logFC	pct.1	pct.2	校正后的P值	pct_FC
Nsg2	5×10^-324	0.77	0.25	0.001	5×10^-324	253.99
Cd8b1	5×10^-324	1.89	0.49	0.012	5×10^-324	40.42
Cd8a	5×10^-324	1.67	0.44	0.014	5×10^-324	31.36
Tcf7	5×10^-324	1.95	0.65	0.027	5×10^-324	24.22
Sidt1	1.63×10^-303	0.92	0.31	0.013	3.89×10^-299	24.15
Cd5	4.64×10^-292	1.06	0.32	0.016	1.11×10^-287	19.99
Lef1	5×10^-324	1.24	0.42	0.025	5×10^-324	16.64
Themis	3.21×10^-241	0.84	0.30	0.018	7.66×10^-237	16.44
Cd247	1.03×10^-282	0.91	0.35	0.022	2.44×10^-278	15.99
Bcl11b	5×10^-324	1.57	0.60	0.039	5×10^-324	15.44
注：avg_logFC，平均对数倍数变化； pct.1，在第6簇的表达百分比；pct.2，在其他细胞簇的表达百分比； pct-FC，百分比倍数变化。

下载: 导出CSV

表 2 第7簇T淋巴细胞前10位特异性基因

Table 2. Top10 specific DEGs of Cluster 7

基因	P值	avg_logFC	pct.1	pct.2	校正后的P值	pct_FC
Cd40lg	7.69×10^-294	0.73	0.26	0.006	1.83×10^-289	42.50
Tcrg-C1	5×10^-324	1.56	0.46	0.013	5×10^-324	35.00
Il2ra	5×10^-324	0.99	0.34	0.011	5×10^-324	31.00
Cxcr6	5×10^-324	2.32	0.90	0.04	5×10^-324	22.50
Klrb1c	5×10^-324	1.53	0.58	0.03	5×10^-324	19.47
Tcrg-C2	5×10^-324	1.48	0.44	0.023	5×10^-324	19.04
Prrt1	3.39×10^-258	0.87	0.32	0.017	8.09×10^-254	18.76
Tcrg-C4	7.30×10^-290	1.16	0.37	0.02	1.74×10^-285	18.50
Il12rb2	9.05×10^-193	0.64	0.25	0.014	2.16×10^-188	17.93
Cd160	5×10^-324	1.12	0.45	0.025	5×10^-324	17.80
注：pct.1，在第7簇的表达百分比；pct.2，在其他细胞簇的表达百分比。

下载: 导出CSV

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