采用单细胞测序技术探索博来霉素诱导的肺纤维化中 Treg 细胞的变化情况

Exploration on changes of Treg cells in bleomycin induced pulmonary fibrosis by single cell sequencing technology

关键词: 单细胞测序  调节性 T 细胞(Treg cells)  肺纤维化

Keywords: single cell sequencing  regulatory T cells (Treg cells)  pulmonary fibrosis

摘 要: 目的 利用单细胞测序技术探讨博来霉素诱导的肺纤维化模型中调节性 T(Treg)细胞的动态变化及其作用。方法 选取 24 只雌性 SPF 级 C57BL/6 小鼠,随机分为空白对照组、博来霉素模型 14d 组和 28d 组;模型组小鼠气管内注射 100μl 博来霉素溶液,对照组以相同方法注射等量的氯化钠注射液;造模完成后处死小鼠,分离肺组织并制备单细胞悬液。通过单细胞测序分析识别并观察 Treg 细胞的数量变化及差异基因表达情况,利用 DAVID 数据库对关键差异基因进行 GO 生物学功能和 KEGG 信号通路富集分析,最后通过细胞通讯分析评估 Treg 细胞与其他 T 细胞亚群之间的相互作用。结果 单细胞测序结果显示,在造模 14d 时 Treg 细胞比例增加,而在 28d 时反显著下降。功能分析表明, Treg 细胞在造模后 14d 的富集主要见于白细胞分化、淋巴及单核细胞增殖、Th17 细胞以及 Th1/Th2 细胞分化等过程;在 28d 时的富集则主要见于白细胞凋亡、细胞外基质(ECM)-受体相互作用、肌动蛋白细胞骨架调节和 Toll 样受体信号传递等环节。细胞通讯分析显示,Treg 细胞与 Th1 细胞之间的相互作用最为显著。结论 在肺纤维化早期,Treg 细胞可能通过增强上游炎症反应促进疾病进展;而在晚期,Treg 细胞可能通过分泌促纤维化细胞因子加重病情,其耗竭可能导致 Th1 向 Th2 转化,从而进一步加剧疾病进程。

Abstract: Objective Explore the dynamic changes and role of regulatory T cells(Treg cells) in bleomycin induced lung fibrosis model using single cell sequencing technology. Methods Twenty-four female SPF grade C57BL/6 mice were randomly divided into three groups: blank control group,bleomycin modelling group A and bleomycin modelling group B;the modelling mice of A or B group were given 100 μl of bleomycin solution by trachea per day executed for 14 or 28 days, respectively,the control rats were endotracheally injected with equal amount of sodium chloride, the rats were killed and the lung tissues were isolated for preparing of single cell suspension. Then,identify Treg cells and observe the changes in their number and differential gene expression by single cell sequencing analysis,then the GO biological function and KEGG signaling pathway enrichment analysis on key differentially expressed genes was performed using the DAVID database, finally,the interactions between Treg cells and other T cell subpopulations were assessed through cell communication analysis. Results The single cell sequencing showed that the proportion of Treg cells was increased at 14 days after modelling but decreased significantly at 28 days,functional analysis showed that the enrichment of Treg cells at 14th day after modelling was mainly found in the links of leukocyte differentiation, lymphocyte and monocyte proliferation,Th17 cell and Th1/Th2 cell differentiation;at the 28th day after modelling,the enrichment was mainly found with such processes as leukocyte apoptosis,ECM-receptor interactions,actin cytoskeleton regulation,and Toll like receptor signaling,etc.The analysis of cellular communication showed that the interaction between Treg cells and Th1 cells was the most significant. Conclusion The results suggested that in the early stages of pulmonary fibrosis,Treg cells may promote disease progression by enhancing upstream inflammatory responses,whereas in late stage,Treg cells may exacerbate the condition of disease by secreting pro-fibrotic cytokines,its depletion may lead to the conversion of Th1 to Th2,which may further exacerbate the disease process.