缺血性脑卒中后小鼠脑组织中星形胶质细胞的异质性分析

Heterogeneity of astrocytes in mouse brain tissue after ischemic stroke

目的 基于单细胞测序技术对缺血性脑卒中(IS)后脑组织中星形胶质细胞的动态变化进行研究,以更好地理解星形胶质细胞在IS发生、发展中的作用。方法 利用基因表达汇编(GEO)数据库下载IS小鼠脑组织测序数据(GSE227651),采用典型关联分析(CCA)法进行数据整合,通过t-分布随机近邻嵌入(tSNE)聚类降维分析获取不同的细胞亚群,使用SingleR包对不同细胞亚群进行注释;进一步通过tSNE聚类降维分析获取不同的星形胶质细胞亚群,并对不同星形胶质细胞亚群的数量改变和功能状态进行分析;利用Monocle包对不同星形胶质细胞亚群所处的发育阶段进行分析;利用CellChat包对星形胶质细胞与其他细胞亚群之间的配体-受体相互作用情况进行动态分析。结果 下载获得GSE227651测序数据,整合聚类分析后将小鼠脑组织细胞聚类为19个细胞亚群,注释为16种不同的细胞类型。进一步对星形胶质细胞进行聚类分析,分为6个星形胶质细胞亚群,其中亚群0和亚群3在IS后第1天的数目占比相较于健侧对照降低,在IS后第3和第7天的占比逐渐升高;而亚群2和亚群5在IS后第1天的数目占比相较于健侧对照升高,在IS后第3和第7天的占比逐渐降低;亚群1和亚群4在IS后不同时间点的变化不大。根据功能分析将星形胶质细胞亚群2和亚群5定义为反应性星形胶质细胞,亚群0和亚群3定义为修复性星形胶质细胞,亚群1和亚群4定义为静息性星形胶质细胞。根据拟时序分析结果进一步将星形胶质细胞亚群2定义为急性反应性星形胶质细胞,亚群0定义为缺血损伤后修复性星形胶质细胞。细胞配体-受体相互作用分析显示,星形胶质细胞与星形胶质细胞、B细胞、自然杀伤(NK)细胞、内皮细胞、巨噬细胞、上皮细胞和神经元的配体-受体作用关系随时间动态改变,在IS后第1天主要通过与NK细胞建立联系介导炎症反应,而在IS后第3天开始通过与内皮细胞和神经元联系介导神经血管功能的恢复。结论 星形胶质细胞的功能状态在IS后随时间动态改变,在IS后的早期可能主要通过急性反应性星形胶质细胞介导损伤后的急性炎症反应,而从IS后第3天开始可能主要通过缺血损伤后修复性星形胶质细胞参与损伤后脑组织的功能重建。

Objective To study the dynamic changes of astrocytes in brain tissue after ischemic stroke(IS)by single cell sequencing technology,so as to better understand the role of astrocytes in the development and progression of IS.Methods The sequencing data of IS mouse brain tissue(GSE227651)were downloaded from the Gene Expression Omnibus(GEO)database,and the canonical correlation analysis(CCA)method was used for data integration.Different cell subsets were obtained by t-distributed stochastic neighbor embedding(tSNE)dimensionality reduction and cluster analysis,and different cell subsets were annotated by SingleR package.Further,different astrocyte subsets were obtained by tSNE dimensionality reduction and cluster analysis,and the number change and functional status of different astrocyte subsets were analyzed.Monocle package was used to analyze the developmental stages of different astrocyte subsets.Dynamic analysis of ligand-receptor interactions between astrocytes and other cell subsets was performed using CellChat package.Results The sequencing data of GSE227651 were downloaded and the mouse brain cells were grouped into 19 cell subsets and annotated into 16 different cell types after integrated cluster analysis.The astrocytes were further divided into 6 astrocyte st subsets by cluster analysis.Compared with the healthy control,the number of subsets 0 and 3 decreased on the 1 day after rd th st IS,but gradually increased on the 3 and 7 day after IS;the number of subsets 2 and 5 increased on the 1 day after IS,and rd th gradually decreased on the 3 and 7 day after IS;subsets 1 and 4 had little change at different time points after IS.Based on functional analysis,astrocyte subsets 2 and 5 were defined as reactive astrocytes,subsets 0 and 3 were defined as reparative astrocytes,and subsets 1 and 4 were defined as resting astrocytes.According to the results of pseudotime analysis,astrocyte subset 2 was further defined as acute reactive astrocytes,and subset 0 was defined as reparative astrocytes after ischemic injury.The analysis of cell ligand-receptor interactions showed that the ligand-receptor interactions between astrocytes and astrocytes,B cells,natural killer(NK)cells,endothelial cells,macrophages,epithelial cells and neurons changed dynamically st with time,and the inflammatory response was mediated mainly by establishing association with NK cells on the 1 day after rd IS.The recovery of neurovascular function was mediated by the connection with endothelial cells and neurons from the 3 day of IS.Conclusion The functional status of astrocytes changes dynamically with time after IS.In the early period after IS,rd acute reactive astrocytes may mediate the acute inflammatory response after injury,while from the 3 day after IS,reparative astrocytes may participate in the functional reconstruction of brain tissue after injury.