基于单细胞测序发现心脏再灌注损伤中存在一群Fabp5+巨噬细胞新亚群

Fabp5+ macrophage subpopulation was revealed during cardiac reperfusion injury by single-cell sequencing

目的:应用单细胞测序技术,揭示小鼠心脏在缺血再灌注后浸润的巨噬细胞中存在新亚群。方法:选取C57小鼠10只以及绿色荧光蛋白(GFP)基因小鼠,通过伽马射线对C57小鼠进行照射后构建移植有GFP阳性骨髓的小鼠;选用骨髓移植成功的小鼠通过冠脉前降支结扎30 min后再恢复灌注构建心脏缺血再灌注(I/R)模型;于术后3 d处死小鼠并收集新鲜心脏组织,采用美天旎心脏组织消化试剂盒获得细胞悬液,实验分为对照组:单纯利用经典流式细胞分选技术分析I/R 3 d时心脏中的骨髓源性巨噬细胞;实验组:在流式分选的基础上通过新方法C1微流控技术进行单细胞捕获以及转录组学测序。结果:首先通过流式分选技术我们发现心脏中存在两群巨噬细胞:大量骨髓源性的巨噬细胞(CCR2+GFP+,约占80%),以及CCR2-的原位巨噬细胞(约占20%);进一步捕获单个的CCR2+GFP+细胞并构建单细胞的cDNA文库;结合测序数据饱和度以及内部对照spike-in RNA对数据进行筛选得到47个细胞的数据;经过分析发现存在两个细胞亚群,其中一群巨噬细胞高表达Fabp5。结论:通过单细胞测序发现心脏缺血再灌注过程中存在一群新的Fabp5阳性的骨髓来源的巨噬细胞亚群。

Objective: Reveal the subpopulations of macrophages infiltrated after ischemia-reperfusion(I/R) in the mouse heart by single-cell sequencing. Methods: Ten C57 mice and green fluorescent protein(GFP+) mice were selected, and C57 mice were transplanted with GFP-positive bone marrow after irradiated with gamma rays;Mice with successful bone marrow transplantation were selected to construct the I/R model through ligation of the anterior descending coronary artery and reperfusion was resumed thirty minutes later. The mice were sacrificed three days later and the cell suspension of fresh heart tissue was obtained using Miltenyi Heart Tissue Digestion Kit. Then divided into a control and experimental group: purely classical flow cytometry sorting technology analyzes the bone marrow-derived macrophages as control and combined with single-cell transcriptomic sequencing using the new method C1 microfluidic technology as the experiment group. Results: First, through flow sorting, we found there were two groups of macrophages in the heart: a large number of bone marrow-derived macrophages(CCR2+GFP+;accounting for about 80%), and CCR2-in situ macrophages(approximately 20%). Then, individual CCR2+GFP+ macrophage was captured and single-cell cDNA library was constructed, combined with sequencing data saturation and internal control spike-in RNA were used to screen the data and we obtained qualified data of 47 cells. Two cell subpopulations were revealed and one group of macrophages with high expression of Fabp5. Conclusions: Through single-cell sequencing, we found a new Fabp5-positive bone marrow-derived macrophage subpopulation during cardiac ischemia-reperfusion.