Study on the characteristics of mononuclear phagocyte subsets after sciatic nerve injury in rats based on single cell sequencing technology
10.3760/cma.j.cn441206-20240208-00044
- VernacularTitle:基于单细胞测序技术分析大鼠坐骨神经损伤后单核吞噬细胞亚群特点的相关研究
- Author:
Shuai FENG
1
;
Zhenjun XIE
;
Jinsheng HUANG
;
Guohong ZHAO
;
Nan ZHOU
Author Information
1. 河南省人民医院（郑州大学人民医院）手足显微与创面修复外科，郑州 450003
- Keywords:
Sciatic nerve injury;
Single-cell RNA sequencing;
Mononuclear phagocytes;
Cell atlas;
Rat
- From:
Chinese Journal of Microsurgery
2024;47(3):312-320
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To reveal the molecular characteristics of mononuclear phagocytes (MPs) in rat model of peripheral nerve injury (PNI) using single-cell RNA sequencing (scRNA-seq) technology that would provide the developmental changes and major biological process involved in the function of MPs after PNI.Methods:Twenty-seven male SD rats (200-300 g in weight) were selected from the Department of Hand and Foot Microscopy and Wound Repair Surgery, Henan Provincial People's Hospital (People's Hospital of Zhengzhou University) and the Department of Orthopaedics of First Affiliated Hospital of Zhengzhou University from July 2023 to December 2023. The rats were divided into a Sham operation group (Sham group), a 3 days post crush group (3 dpc group) and a 7 days post crush group (7 dpc group), following the randomised table method with 9 rats per group. After 7 days of environmental acclimatisation, the 3 dpc group and 7 dpc group were subjected to have the right sciatic nerve crushed in order to create a model of crush injury. And as a control group, the Sham group was subjected to Sham surgery only. Nine right sciatic nerves of rats were collected from each group at the corresponding time pints. Single-cell isolation was performed on the 10X Genomics platform. ScRNA-seq libraries were constructed using the Gel Bead Kit V3 and the libraries were sequenced using an Illumina Novaseq 6000 sequencer. Dimensionality reduction was performed using Principal Component Analysis and T-Distributed Stochastic Neighbor Embedding to visualise and explore the cellular heterogeneity within the dataset. Nine distinct cell clusters and their corresponding marker genes were identified based on the dimensionality-reduced data. Differential gene expression analysis was then performed to identify differentially expressed genes (DEGs) in MPs between different groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to uncover the biological processes and pathways based on the DEGs. Monocle program for pseudo-time analysis was used to infer the developmental trajectory of MPs after injury.Results:A total of 19 054 cells were obtained by sequencing, and the results showed that the proportion of MPs in peripheral nerves was significantly up-regulated after PNI, and MPs were classified into 9 cellular subgroups based on the clustering analysis of the scRNA-seq data, which were Cluster 1 (3 398 cells), Cluster 2 (3 388 cells), Cluster 3 (3 262 cells), Cluster 4 (2 825 cells), Cluster 5 (2 753 cells), Cluster 6 (1 894 cells), Cluster 7 (648 cells), Cluster 8 (492 cells) and Cluster 9 (394 cells), respectively. Based on the expression of different cell subpopulation markers, MPs in the Sham group, 3 dpc group and 7 dpc group of sciatic nerves were classified into 9 cell clusters and the distributions of different MPs clusters in the 9 sciatic nerve samples were identified, among which, the Sham group had the lowest number of MPs cells in the sciatic nerve samples (a total of 2 719 cells) and the clusters were mainly dominated by clusters 5 (1 119 cells) and clusters 6 (1 240 cells). The 3 dpc group had the highest number of MPs cells (9 760 cells in total) and the clusters were mainly dominated by cluster 2 (1 760 cells), cluster 3 (3 130 cells) and cluster 4 (2 300 cells). The MPs (6 575 cells in total) in the 7 dpc group were mainly dominated by cluster 1 (2 406 cells) and cluster 2 (1 628 cells). Compared with the Sham group, the GO and KEGG annotations of the DEGs were significantly upregulated in the 3 dpc group, indicating that MPs in the rat sciatic nerves would have the ability to bind to extracellular molecules and remove debris from the injury site at 3 days post-injury, and the 7 dpc group would have the ability to activate the signalling pathways related to nerve repair. The proposed time-series analysis revealed that, in the uninjured condition, the MPs were mainly in the cluster 5 (Ccl17 +Cd80 +) and cluster 6 (Fcmr +Slc9a9 +). At 3 days post-injury, MPs developed into cell types dominated by cluster 2 (Cd8b +Meis3 +), cluster 3 (Il10 +Cd163 +) and cluster 4 (Ccl24 +Prg4 +). At 7 days post-injury, the effector state of cluster 2 among the main cell types of MPs was still maintained but the other parts had developed into cluster 1 (Hspa1b +Apobec1 +) related phenotypes. Conclusion:The molecular characteristics of MPs in the peripheral nerve revealed through scRNA-seq data provide valuable insights into the role of MPs in mediating inflammation and neural regeneration after PNI.
