Single-cell RNA-seq of vascular endothelial cells in the mouse model of vascular injury

To elucidate the mechanism of vascular repair, we performed single-cell analysis using the mouse vascular injury model (oxygen-induced retinopathy). In this model, mice were exposed to hyperoxic conditions at postnatal day 7 (P7) to induce ischemic retinopathy and the shedding of blood vessels. A return to normal oxygen conditions (normoxia) at P12 results in neovascularization that peaks at P17 and returns to normal by P30. CD31+/CD45neg vascular endothelial cells (ECs) were harvested at P12 (ischemic phase), P17 (retinopathy phase), and P25 (vascular repair phase) from retina only or retina plus optic nerve (6 samples) and analyzed together with corresponding control samples (total of 12 samples). Isolated tissues were digested, stained for CD31/CD45, and isolated by fluorescence-activated cell sorting (FACS). ECs were then loaded into the 10x Chromium Single Cell 3' Kit (v3) to obtain approximately 5,000 cells per well.为阐明血管修复的分子机制，本研究采用氧诱导视网膜病变（oxygen-induced retinopathy）小鼠血管损伤模型开展单细胞分析。在该模型中，实验小鼠于出生后第7天（postnatal day 7，P7）暴露于高氧环境，以诱导缺血性视网膜病变及血管脱落；于出生后第12天（P12）恢复至正常氧环境（normoxia）后，会引发新生血管生成，该过程于P17达到峰值，并于P30恢复至正常状态。我们分别于P12（缺血期）、P17（病变期）及P25（血管修复期），从单纯视网膜组织或视网膜加视神经组织中获取CD31+/CD45阴性的血管内皮细胞（vascular endothelial cells，ECs），共获得6份样本，并与对应的对照样本（总计12份样本）联合进行分析。将采集的组织进行消化，用CD31/CD45进行染色，随后通过荧光激活细胞分选术（fluorescence-activated cell sorting，FACS）分离目标细胞；随后将分离得到的内皮细胞加入10x Chromium单细胞3'端测序试剂盒（v3）中，每孔获取约5000个细胞。