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
冯帅 1谢振军 2黄金生 3赵国红 2周南3
作者信息
- 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;郑州大学第一附属医院骨科,郑州 450002
- 2. 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
- 3. 郑州大学第一附属医院骨科,郑州 450002
- 折叠
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19 054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(3 398个细胞)、亚群2(3 388个细胞)、亚群3(3 262个细胞)、亚群4(2 825个细胞)、亚群5(2 753个细胞)、亚群6(1 894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2 719个),且主要以亚群5(1 119个)、亚群6(1 240个)为主;3 dpc组MPs细胞数最多(共9 760个),且主要以亚群2(1 760个)、亚群3(3 130个)、亚群4(2 300个)为主;7 dpc组MPs(共6 575个)以亚群1(2 406个)、亚群2(1 628个)为主;相较于Sham组,3 dpc组中显著上调DEGs的GO和KEGG注释结果显示,大鼠坐骨神经中MPs在损伤后3 d可能具备与胞外分子结合并清除损伤部位碎片的能力,7 dpc组可能具备激活神经修复相关信号通路的能力;拟时序分析揭示了在未损伤时,MPs主要为亚群5(Ccl17+Cd80+)与亚群6(Fcmr+Slc9a9+);损伤后 3 d 时,MPs 发展为以亚群 2(Cd8b+Meis3+)、亚群 3(I110+Cd163+)、亚群 4(Ccl24+Prg4+)为主的细胞类型;损伤后7 d时,MPs主要细胞类型中亚群2的效应状态仍得以保持,但其他部分发展为亚群1(Hspa1b+Apobec1+)相关表型.结论 通过scRNA-seq数据揭示周围神经中MPs的分子特征,有利于深入了解MPs在PNI后介导炎症反应和神经再生中的作用.
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 environ-mental 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 iden-tified 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 Encyclo-pedia 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 high-est 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 clus-ter 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.
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Key words
Sciatic nerve injury/Single-cell RNA sequencing/Mononuclear phagocytes/Cell atlas/Rat引用本文复制引用
基金项目
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)
出版年
2024
NETL
NSTL
万方数据