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

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。

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.

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

Key words

single-cell RNA sequencing / scar-associated macrophage / hepatic stellate cell / liver fibrosis