单细胞RNA测序探索多发性硬化患者外周血中免疫细胞特征

An exploration of immune cell signatures in peripheral blood of multiple sclerosis patients by single-cell RNA sequencing

目的 探讨多发性硬化(Multiple sclerosis,MS)患者外周血中免疫细胞特征。方法 通过GEO(Gene expression omnibus)数据库下载单细胞数据,利用seurat标准程序对数据进行质控、降维及分群后根据临床数据将各类细胞再次分为MS组和健康对照(Health Identify,HI)组,此后对细胞进行类型间和组间的比较分析;在对细胞类型间的转录组变化分析后对差异表达基因进行富集分析明确各细胞类型的功能;进一步对细胞组间基因差异最显著和最多的细胞类型进行亚群分析,比较其上调亚群和下调亚群的转录组变化并进行富集分析。结果 通过对数据标准化和过滤后共获得71592个细胞,经过线性和非线性降维分析后获得21个边界清晰的细胞群,对细胞群注释后将其分为6种细胞类型,包括CD4+T细胞、CD8+T细胞、B细胞、自然杀伤细胞、初始CD8+T细胞、单核细胞-巨噬细胞;转录组的富集分析显示这6种细胞主要与T细胞的活化和受体的激活信号通路相关;亚群的转录组分析显示CD8+T细胞的上调亚群主要与先天性免疫、适应性免疫和NK细胞抑制有关,而CD4+T的亚群主要富集在Ras信号通路相关的T细胞活化过程,并且其亚群中还发现CCL5基因的高表达。结论 MS血液中的基因表达存在明显的细胞类型特异性,并且还发现CD8+T细胞亚群中的高表达基因与NK细胞抑制的通路有关,而在CD4+T细胞的亚群中发现了CCL5基因的早期高表达。

Objective To investigate immune cell signatures in peripheral blood of patients with multiple sclerosis(MS).Methods The single cell data were downloaded from Gene Expression Omnibus(GEO) database. Standard Seurat workflow was performed for quality control, dimensionality reduction and clustering. Then, each cell type was divided into MS orhealthy control groups based on clinical data. Comparisons among cells types, as well as comparison between groups, were conducted. Transcriptomic analysis of changes among different cell types was performed to disclose genes with significantly different expression levels. The specific function of each cell type was identified by enrichment analysis. Subgroup analysis for cell types with the most significant gene expression differences was also performed. Transcriptomo change comparison between up-regulated and down-regulated subgroups, as well as enrichment analysis, was conducted. Results Total 71592 cells were acquired by data standardization and filteration. Those cells were divided into 21 cell clusters through linear and nonlinear dimensionality reduction. The clusters were annotated into six cell types including CD4+T cells, CD8+T cells, B cells, natural killer(NK) cells, na ve CD8+T cells, and mononuclear macrophages. The enrichment anaysis of transcriptomo showed that the six cell typesmostly correlated with activation and receptor-mediated activating pathways of T cells. And the enrichment analysis of transcriptomo in subgroups demonstrated that up-regulation of CD8+T cell subgroups was related to innate immunity, adaptive immunity and inhibition of NK cells. However, the enrichment of CD4+T cell subgroups occurred in Ras associated activating process of T cells. And high expression of CCL5 gene was detected in these subgroups. Conclusion We found the existence of cell type specificity for different gene expression in the blood of MS patients. We also found high expression of genes in CD8+T subgroups was related to inhibition of NK cells, and CCL5 gene was highly expressed in the early stage in CD4+T cell subgroups.