非节段型白癜风患者皮损单细胞转录图谱的初步研究

A single-cell transcriptome atlas of non-segmental vitiligo lesions:a preliminary study

目的采用单细胞RNA测序技术鉴定和区分白癜风皮损真表皮细胞亚群,并研究它们之间的关系。方法2019年9月在杭州市第三人民医院皮肤科门诊收集2例健康人(无免疫及系统性疾病)正常皮肤和2例非节段型稳定期白癜风患者皮损样本,采用10×Genomics单细胞RNA-Seq技术检测,对所有样本的11000个细胞进行单细胞转录组测序。通过Seurat软件分析、筛选和统计细胞亚群。结果对2例正常皮肤基因表达的聚类分析发现包括角质形成细胞、成纤维细胞、神经及黑素细胞、内皮细胞、组织干细胞和以树突细胞及T细胞为主的免疫细胞群。2例白癜风皮损中分化和数量异常的有成纤维细胞和4类角质形成细胞亚群,其中,成纤维细胞占比为0,低于正常皮肤(0.4%),角质形成细胞亚群5、6、10、12占比(8.03%、7.36%、3.52%、0.91%)均显著高于正常皮肤(4.47%、3.53%、2.69%、0.28%,均P<0.01)。上述亚群的角质形成细胞处于细胞分化的末端,同时还具有极显著且特异的标记基因,分析显示,标记基因主要与细胞间相互作用和细胞稳态密切有关,GO和KEGG分析显示,角质形成细胞亚群5、6主要与细胞间连接和细胞黏附及细胞骨架功能相关,角质形成细胞亚群10与细胞稳态紧密相关。结论国内首次报道通过单细胞测序手段研究白癜风皮损的转录表达谱,初步发现4群数量及功能差异的角质形成细胞,提示角质形成细胞亚群的异常分化与功能异常可能影响白癜风的发生发展。

Objective To identify and differentiate cell subsets in the epidermis and dermis of vitiligo skin lesions using single-cell RNA sequencing technology,and to study the relationship between them.Methods Skin samples were collected from 2 healthy people without immune or systemic diseases and 2 patients with stable non-segmental vitiligo in Department of Dermatology in the Third People′s Hospital of Hangzhou in September 2019.Single-cell transcriptome sequencing was performed on 11000 cells in all the skin samples by using 10×Genomics single-cell RNA-Seq technology.Cell subsets were analyzed,screened and counted by using Seurat software.Results Cluster analysis of gene expression in the 2 normal skin tissues revealed several cell subsets,including keratinocytes,fibroblasts,nerve cells and melanocytes,endothelial cells,tissue stem cells,and immune cells mainly consisting of dendritic cells and T cells.In the 2 vitiligo lesions,abnormal differentiation and quantity were observed in fibroblasts and 4 keratinocyte subpopulations.The proportion of fibroblasts was significantly lower in vitiligo lesions than in normal skin tissues(0 vs.0.4%),while the proportions of keratinocyte subpopulations 5,6,10 and 12(8.03%,7.36%,3.52%,0.91%,respectively)in vitiligo lesions were significantly higher than those in the normal skin tissues(4.47%,3.53%,2.69%,0.28%,respectively,all P<0.01).Moreover,the above keratinocyte subpopulations were at the end of cell differentiation,and expressed very significant and specific marker genes,which were mainly closely related to cell-cell interactions and cell homeostasis.GO and KEGG analysis showed that keratinocyte subpopulations 5 and 6 were mainly related to intercellular connection,cell adhesion and cytoskeleton function,while the keratinocyte subpopulation 10 was closely related to cell homeostasis.Conclusion The single-cell sequencing technology was firstly used to study the transcriptional expression profile of vitiligo lesions in China,and preliminary analysis revealed 4 groups of keratinocytes with different quantity and functions,suggesting that abnormal differentiation and dysfunction of keratinocyte subpopulations may affect the occurrence and development of vitiligo.