单细胞测序分析老年小鼠头皮伤口愈合过程中SPP1+巨噬细胞生物行为

Study of biological behavior of SPP1+macrophages during scalp wound healing in aged mice using single⁃cell sequencing

目的:通过单细胞测序分析老年小鼠头皮伤口愈合过程中SPP1+巨噬细胞在细胞成分、富集通路分析、细胞通讯和基因差异分析等方面与成年小鼠的差异,初步探讨导致老年患者皮肤伤口愈合不良的可能机制。方法:选取不同鼠龄的C57BL/6小鼠构建头皮创伤动物模型,使用流式分选技术获得小鼠皮肤伤口处的炎性细胞。使用Illumina平台Novaseq 6000测序仪进行单细胞测序并进行质控。使用PCA降维后获得了13个聚类的细胞群,基于各细胞标志物数据库对各聚类进行手工细胞注释。选取SPP1+巨噬细胞进行富集通路分析及差异基因分析,使用Cellcall工具包进行细胞通讯分析。结果:实验组和对照组在质控后分别获得2405和8355个细胞数据,分群分析后进行细胞注释发现实验组SPP1+巨噬细胞分布显著高于对照组。实验组SPP1+巨噬细胞显著上调的基因功能主要富集于糖代谢/糖酵解通路,与上皮细胞通讯显著增加,但与CD4+辅助性T细胞17、StageⅡ粒细胞通讯显著减少。基因差异分析显示实验组SPP1+巨噬细胞中多种基因表达升高,包括与衰老密切相关的Mif基因。结论:老年小鼠皮肤伤口内SPP1+巨噬细胞占比增加,糖代谢/糖酵解通路活跃,细胞间通讯变化和Mif表达增加,通过研究老年患者皮肤伤口愈合能力下降的机制,为促进其伤口愈合提供临床治疗基础。

Objective:To analyze the effects of SPP1+macrophages on cellular composition,enriched pathways,cell communication,and gene differential analysis during the healing process of scalp wounds in aged mice using single⁃cell se⁃quencing.To explore the possible mechanisms leading to impaired skin wound healing in elderly patients.Methods:C57BL/6 mice were selected to establish a scalp wound animal model,and inflammatory cells were obtained from the wound site using flow cytometry technique/method.Single⁃cell sequencing was performed using the Illumina Novaseq 6000 platform,followed by quality control measures.After applying principal component analysis(PCA)for dimension reduction,13 clusters of cells were identified.Manual cell annotation was performed based on various cell marker databases.SPP1+macrophages were select⁃ed for enriched pathway analysis and differential gene analysis,and cell communication analysis was conducted using the Cellcall toolkit.Results:After quality control,the experimental and control groups obtained 2405 and 8355 cell data respec⁃tively.Cell annotation following cluster analysis revealed a significantly higher distribution of SPP1+macrophages in the exper⁃imental group compared to that in the control group.Functionally,the upregulated genes in SPP1+macrophages of the experi⁃mental group were mainly enriched in glycolysis/glycolytic pathways,and the communication between SPP1+macrophages and epithelial cells was significantly increased,while the communication between SPP1+macrophages and CD4+helper T cells 17 and StageⅡgranulocytes was significantly decreased.Gene differential analysis showed elevated expression of multi⁃ple genes in SPP1+macrophages of the experimental group,including Mif gene,which is closely associated with aging.Con⁃clusion:Aging may increase the number of SPP1+macrophages in skin wounds,leading to increased activity in glycolysis/glycolytic pathways,altered cell communication,and increased expression of Mif.By studying the mechanisms underlying the decreased wound healing capacity in elder,to a clinical basis for promoting wound healing.