摘要： 目的 研究皮肤自然衰老过程中基因表达和免疫微环境的动态变化。方法 收集GTEx项目中699个皮肤组织转录组测序数据（RNA-seq），依据年龄段分为年轻组（20~39岁）和衰老组（60~79岁），进行差异基因表达分析。用CIBERSORT预测样本的免疫细胞浸润度，分析其随皮肤衰老的动态变化；用Seurat对已公开皮肤单细胞转录组数据（scRNA-seq）进行分析，观察免疫细胞群体随衰老的动态变化；比较年轻组和衰老组各细胞类群中免疫相关基因的动态表达变化。结果 对年轻组与衰老组进行差异表达分析显示，非暴露和暴露皮肤组织中转录表达水平发生了明显变化，衰老皮肤组织中转录上调的基因中显著富集炎症相关通路；多年龄段数据分析显示49个炎症水平相关基因的表达水平随皮肤衰老逐渐上调。免疫细胞浸润度预测分析显示年轻组与衰老组间无明显差异，但其在衰老皮肤组织中个体异质性大；对衰老和年轻皮肤组织的scRNA-seq进行分析，发现T细胞及其他免疫细胞亚群的大小随衰老逐渐下降，同时发现非免疫细胞中免疫相关基因随皮肤衰老表达上调。结论 皮肤衰老过程中基因转录表达和免疫微环境发生明显改变，炎症相关基因明显上调；T细胞类群数量明显减少,多种主要非免疫细胞类群表达炎症相关因子，表明非免疫细胞可能在皮肤衰老过程中发挥重要作用。

关键词: 皮肤, 衰老, 单细胞, 免疫微环境, 转录组学

Abstract: Objective To investigate the changes in transcriptome and immune microenvironment during human skin aging through integrative analysis of multiomics.Methods We collected RNA-seq data from 699 human skin tissues in GTEx project. Then, we analyzed gene expression in young (20~39) and aged (60~90) group using RNA-seq datasets. CIBERSORT were used to calculate the immune infiltration of each sample and analyzed the dynamics of immune infiltration during skin aging. The dynamics of immune cell population in the aged and young skin were compared through integrative analysis of singlecell RNA-seq derived from 2 young and 3 old subjects using Seuate R package. In addition, we investigated the expression of immunerelated genes in immune cells and nonimmune cells of the skin. Results Distinct transcriptomic differences were observed between aged and young group in both nonsun-exposed and sun-exposed skin. The up-regulated genes in aged group were enriched in inflammatory pathway. Particularly, 49 inflammatoryrelated genes were gradually up-regulated during skin aging. Even though we didn′t observe the significant changes in immune cell infiltration between aged and young group, a heterogeneity of the immune cell infiltration was observed in aged group. Further, scRNA-seq analysis showed that CD4⁺/CD8⁺ T cells were reduced in the aged skin, indicating the declined adaptive immune ability in the aged skin. Interestingly, we found that some nonimmune cell types expressed immune-related genes, suggesting that they may contribute to the increased inflammation in the aged skin. Conclusions  The cutaneous microenvironment and transcriptome change with aging, with elevated expression of inflammationrelated genes. In comparison to the young skin, the aged skin displays reductions in T cells and increased expression levels of inflammationrelated genes in non-immune cells, indicating a crucial role of nonimmune cells in skin aging.

Key words: skin, aging, single cell RNAseq, immune microenvironment, transcriptomic