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  

Study on the characteristics of mononuclear phagocyte subsets after sciatic nerve injury in rats based on single cell sequencing technology

作　　者：冯帅 谢振军 黄金生 赵国红 周南 FENG Shuai;XIE Zhenjun;HUANG Jinsheng;ZHAO Guohong;ZHOU Nan(Department of Hand and Foot Microscopy and Wound Repair Surgery,Henan Provincial Hospital(People's Hospital of Zhengzhou University),Zhengzhou 450003,China;Department of Orthopaedics,the First Afiliated Hospital of ZhengzhouUniversity,Zhengzhou450002,China)

机构地区：[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 [2]郑州大学第一附属医院骨科,郑州450002

出　　处：《中华显微外科杂志》2024年第3期312-320,共9页Chinese Journal of Microsurgery

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

摘　　要：目的通过单细胞转录组测序(scRNA-seq)技术探讨大鼠周围神经损伤(PNI)模型中单核吞噬细胞(MPs)的分子特征,揭示MPs在PNI后的细胞亚群发展变化及相关的生物学过程。方法2023年7月至2023年12月于河南省人民医院(郑州大学人民医院)手足显微与创面修复外科及郑州大学第一附属医院骨科选取雄性SD大鼠(体质量200~300 g)27只,根据随机表法将大鼠分为假手术(Sham)组、挤压伤后3 d(3 dpc)组和挤压伤后7 d(7 dpc)组,每组各9只。经7 d环境适应后,对3 dpc和7 dpc组予以右侧坐骨神经卡压以制造卡压伤模型,Sham组仅予以假手术作为对照组。在相应的分组对应时间收集大鼠右侧坐骨神经各9条,制备单细胞悬液,经10X Genomics平台进行单细胞分离,使用Gel Bead Kit V3构建单细胞RNA-seq文库,Illumina Novaseq 6000测序仪对文库进行测序,并利用主成分分析和T分布随机领域嵌入进行降维,获得9个MPs亚群及对应细胞亚群的标记基因,对不同组MPs差异基因进行GO和KEGG分析以揭示其参与的生物学过程。通过Monocle程序进行拟时序分析以揭示损伤后周围神经中MPs发展轨迹。结果测序中共获取19054个细胞,结果显示PNI后周围神经中MPs比例显著上调,根据scRNA-seq数据聚类分析将MPs分为9个细胞亚群,分别为亚群1(3398个细胞)、亚群2(3388个细胞)、亚群3(3262个细胞)、亚群4(2825个细胞)、亚群5(2753个细胞)、亚群6(1894个细胞)、亚群7(648个细胞)、亚群8(492个细胞)、亚群9(394个细胞);根据不同细胞亚群标记物的表达,将坐骨神经Sham组、3 dpc组和7 dpc组中的MPs划分为9种细胞亚群,并鉴定不同MPs亚群在9例坐骨神经样本中的分布。其中,Sham组坐骨神经样本中的MPs细胞数最少(共2719个),且主要以亚群5(1119个)、亚群6(1240个)为主;3 dpc组MPs细胞数最多(共9760个),且主要以亚群2(1760个)、亚群3(3130个)、亚群4(2300个)为主;7 dpc组MPs(共6575个)以亚群1(ObjectiveTo 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.MethodsTwenty-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

关 键 词：坐骨神经损伤 单细胞转录组测序 单核吞噬细胞 细胞图谱 大鼠 

分 类 号：R745[医药卫生—神经病学与精神病学]