基于单细胞测序的大鼠周围神经损伤后成纤维细胞命运分析  

Single-cell sequencing-based analysis of fibroblast fate after peripheral nerve injury in rats

作　　者：李来好 李森瑞[2] 黄金生 李洪珂 刘帅 杨贤玉 路来金 周南[2] Li Laihao;Li Senrui;Huang Jinsheng;Li Honghe;Liu Shuai;Yang Xianyu;Lu Lajin;Zhou Nan(Department of Spinal Surgery,General Hospital of Pingmei Shenma Group,Pingdingshan 467000,China;Department of Orthopedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China;Department of Hand Surgery,the First Hospital of Jilin University,Changchun 130000,China)

机构地区：[1]平煤神马集团总医院脊柱外科,平顶山467000 [2]郑州大学第一附属医院骨科,郑州450000 [3]吉林大学第一医院手外科,长春130000

出　　处：《中华手外科杂志》2025年第1期68-75,共8页Chinese Journal of Hand Surgery

基　　金：国家自然科学基金(82071388);河南省优秀青年科学基金(212300410077);河南省科技开发计划联合基金(232301420063)。

摘　　要：目的:通过单细胞转录组测序(single-cell RNA sequencing,scRNA-seq)技术分析大鼠周围神经损伤(peripheral nerve injury,PNI)模型中成纤维细胞(fibroblasts,FBs)的分子特征,揭示其在PNI后细胞亚群及细胞命运变化。方法:根据随机表法将18只SD大鼠分为对照组(WT)和挤压伤后7 d损伤组(PNI),每组各9只。经7 d环境适应后,PNI组行右侧坐骨神经挤压伤,WT组仅行假手术。分别收集大鼠右侧坐骨神经各9根,制备单细胞悬液,使用GEXSCOPE?单细胞测序试剂盒构建单细胞RNA-seq文库,细胞大类分群后将FBs进一步聚类,获得8个FBs亚群及对应标记基因,对不同组FBs差异基因进行功能富集分析;通过CytoTrace和UCell预测8个FBs亚群分化状态和功能;通过Monocle程序进行拟时序分析以揭示损伤后周围神经中FBs发展轨迹。结果:测序共获取76389个细胞,通过注释得到26919个FBs,PNI后坐骨神经中FBs占比显著下降;根据聚类分群将FBs分为8个细胞亚群(Fibroblast 1~8),通过UCell基因集评分对8个亚群进行评估,以推测其来源和功能;CytoTrace显示分化程度从低到高分别为Fibroblast 8、Fibroblast 7、Fibroblast 3、Fibroblast 2、Fibroblast 4、Fibroblast 5、Fibroblast 1和Fibroblast 6;拟时序分析提示周围神经FBs主要存在两个分化轨迹,推测其与FBs来源有关;稳态时坐骨神经FBs细胞成熟度较高,PNI后干性较强FBs亚群Fibroblast 8和Fibroblast 7丰度明显增加,以进行增殖和修复。结论:scRNA-seq揭示了大鼠周围神经损伤前后FBs的基因表达和各亚型占比存在显著差异。PNI后FBs出现增殖,多类亚群可能分别参与炎症调控、上皮重塑和促血管再生等生物学活动并参与神经再生过程。对FBs各亚群的进一步探究有助于深入了解FBs在PNI过程中细胞命运变化及其作用。Objective:To analyze the molecular characteristics of fibroblasts(FBs)in a rat peripheral nerve injury(PNI)model by single cell RNA sequencing(scRNA seq)technology,revealing changes in cell subpopulations and cell fate after PNI.Methods:Eighteen SD rats were randomly divided into a control group(WT)and a 7-day post crush injury group(PNI)using a random table method,with 9 rats in each group.After 7 days of environmental adaptation,the PNI group underwent right sciatic nerve compression injury,while the WT group only underwent sham surgery.Nine right sciatic nerves were collected from rats separately,single-cell suspension was prepared,and GEXSCOPE?single-cell sequencing kit was used to construct single-cell RNA seq library.After grouping the cells into large groups,FBs were further clustered to obtain 8 FBs subgroups and corresponding marker genes.The functional enrichment analysis was performed on the differentially expressed genes in different groups of FBs.The differentiation status and function of 8 FBs subgroups were predicted using CytoRace and UCell.The proposed chronological analysis was carried out by the Monocle program to reveal the trajectory of the development of the FBs after PNI.Results:A total of 76389 cells were obtained through sequencing,and 26919 FBs were annotated.After PNI,the proportion of FBs in the sciatic nerve significantly decreased.FBs were divided into 8 cell subgroups(Fibroblast 1 to 8)based on clustering,and 8 cell subgroups were evaluated using the UCell gene set score to infer their origins and functions.CytoTrace showed that the differentiation levels from low to high were Fibroblast 8,Fibroblast 7,Fibroblast 3,Fibroblast 2,Fibroblast 4,Fibroblast 5,Fibroblast 1,and Fibroblast 6,respectively.The proposed temporal analysis suggested that there were two main differentiation trajectories of peripheral nerve FBs,which were speculated to be related to the origin of FBs.At steady state,the maturity of sciatic nerve FBs cells was relatively high,and after PNI,the abundance of strong dr

关 键 词：周围神经 成纤维细胞 单细胞转录组测序 细胞命运 

分 类 号：R651.3[医药卫生—外科学]