通过单细胞测序分析瘢痕相关巨噬细胞影响肝星状细胞活化的分子机制

doi:10.3969/j.issn.1006-7795.2024.02.015

首都医科大学学报 ›› 2024, Vol. 45 ›› Issue (2): 271-280.doi: 10.3969/j.issn.1006-7795.2024.02.015

通过单细胞测序分析瘢痕相关巨噬细胞影响肝星状细胞活化的分子机制

刘畅，李丽英，常娜^*

首都医科大学细胞生物学系，‘肝脏保护和再生调节’北京市重点实验室，北京 100069

收稿日期:2023-11-13 出版日期:2024-04-21 发布日期:2024-04-25
通讯作者: 常娜 E-mail:changna@ccmu.edu.cn
基金资助:
国家自然科学基金项目（81770603）。

Single-cell RNA sequencing analyses the mechanism of scar-associated macrophages promoting hepatic stellate cell activation

Liu Chang, Li Liying, Chang Na*

Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China

Received:2023-11-13 Online:2024-04-21 Published:2024-04-25
Supported by:
This study was supported by National Natural Science Foundation of China (81770603).

摘要/Abstract

摘要： 目的建立体外诱导人巨噬细胞为瘢痕相关巨噬细胞（scar-associated macrophages，SAMs）的实验方案，研究SAMs使肝星状细胞（hepatic stellate cells，HSCs）活化的分子机制。方法利用已发表的人和小鼠纤维化肝组织非实质细胞的单细胞测序数据，分析SAMs中纤维化相关功能基因的表达。使用佛波醇12-十四酸酯13-乙酸酯（phorbol-12-myristate-13-acetate，PMA）和纤维蛋白溶酶原（plasminogen，PLG）把人单核细胞系THP-1诱导成为SAMs。收集SAMs培养上清与人HSCs细胞系LX-2共培养。采用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）的方法检测SAMs标志物、纤维化相关功能基因和HSCs活化标志物的表达。结果单细胞测序结果显示，不同病因造成的小鼠纤维化肝组织或肥胖患者损伤肝组织中均存在SAMs，且均高表达其标志基因和纤维化相关基因SPP1、CTSD。联合使用PMA及PLG使THP-1的SAMs标志物CD9、TRME2、LGALS3、CD63表达水平升高，同时SPP1、CTSD表达水平升高。体外诱导的SAMs培养上清可以使LX-2活化。结论 SAMs存在于各种病因导致的人或小鼠损伤/纤维化肝组织中，并具有相似的特征和功能。联合使用PMA及PLG可将THP-1诱导为SAMs。SAMs可能通过产生SPP1、CTSD促进肝星状细胞活化，从而推进肝纤维化的发生与发展。

关键词: 单细胞测序, 瘢痕相关巨噬细胞, 肝星状细胞, 肝纤维化

Abstract: Objective To establish an experimental protocol for inducing human macrophages into scar-associated macrophages (SAMs) in vitro. To investigate the mechanism underlying SAMs regulating hepatic stellate cell (HSC) activation. Methods Published single-cell RNA sequencing (scRNA-Seq) data, which were obtained from human or murine fibrotic liver tissues, were used to study the expression of fibrosis-related genes in SAMs. Phorbol-12-myristate-13-acetate (PMA) and plasminogen (PLG) were used to induce the transformation of human monocyte THP-1 into SAMs. SAM supernatant was collected to culture with LX-2. The expressions of SAM markers, fibrosis-related genes and HSC activation markers were detected by real-time quantitative polymerase chain reaction（RT-qPCR）. Results scRNA-seq revealed that SAMs were detected in both mouse fibrotic livers or human injured liver. SAMs highly expressed fibrosis-related genes such as SPP1, CTSD. PMA and PLG increased the expressions of SAM markers CD9, TRME2, LGALS3, CD63 in THP-1. Fibrosis-related genes SPP1, CTSD were highly expressed by SAMs. The supernatant of SAMs activated LX-2. Conclusion SAMs are accumulated in mouse or human liver injury/fibrosis caused by different causes, and represent similar characteristics and functions. PMA and PLG induce the transformation of human monocyte THP-1 into SAMs. SAMs promote the activation of HSCs via SPP1 and CTSD, thus promoting the occurrence and development of liver fibrosis.

Key words: single-cell RNA sequencing, scar-associated macrophage, hepatic stellate cell, liver fibrosis

中图分类号:

R575.2

刘畅, 李丽英, 常娜. 通过单细胞测序分析瘢痕相关巨噬细胞影响肝星状细胞活化的分子机制[J]. 首都医科大学学报, 2024, 45(2): 271-280.

iu Chang, Li Liying, Chang Na. Single-cell RNA sequencing analyses the mechanism of scar-associated macrophages promoting hepatic stellate cell activation[J]. Journal of Capital Medical University, 2024, 45(2): 271-280.

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通过单细胞测序分析瘢痕相关巨噬细胞影响肝星状细胞活化的分子机制

Single-cell RNA sequencing analyses the mechanism of scar-associated macrophages promoting hepatic stellate cell activation

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