基于单细胞转录组测序探讨小鼠脉络膜新生血管内皮细胞的病理特征  

Investigation of endothelial cell pathological characteristics in murine choroidal neovascularization model based on single-cell RNA sequencing

作　　者：温立势 张泉 严宏祥 李曼红 苏静波 常天芳 王雨生 孙嘉星 Wen Lishi;Zhang Quan;Yan Hongxiang;Li Manhong;Su Jingbo;Chang Tianfang;Wang Yusheng;Sun Jiaxing(Department of Ophthalmology,Xijing Hospital,Air Force Military Medical University,Eye Institute of Chinese PLA,Xi'an 710032,China;Cadet Team 6 of School of Basic Medicine,Air Force Military Medical University,Xi'an 710032,China;the Department of Neurobiology,School of Basic Medicine,Air Force Military Medical University,Xi'an 710032,China)

机构地区：[1]空军军医大学西京医院眼科、全军眼科研究所,西安710032 [2]空军军医大学基础医学院学员六大队,西安710032 [3]空军军医大学基础医学院神经生物学教研室,西安710032

出　　处：《中华实验眼科杂志》2023年第3期241-252,共12页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(81900870、81770936、82000905);中国博士后科学基金项目(2021MD703956);陕西省高校科协青年人才托举计划项目(20220305)

摘　　要：目的基于单细胞转录组测序(scRNA-seq)探讨小鼠脉络膜新生血管(CNV)中内皮细胞(EC)的分子表达与病理特征。方法取6只6~8周龄C57BL/6小鼠按照随机数字表法随机分为2个组,每组3只,处死后摘取双眼眼球,分别采用剪碎脉络膜巩膜复合体法和刮取脉络膜法分离脉络膜组织,采用胰蛋白酶/Ⅰ型胶原酶37℃连续消化制得单细胞悬液,流式细胞术检测细胞活率及EC比例,确定单细胞悬液制备方法。选取6只小鼠随机分为正常对照组和CNV组,每组3只,其中CNV组小鼠建立激光诱导的CNV模型;于造模后7 d制备单细胞悬液,采用10xGenomics进行单细胞分离并构建测序文库,Illumina Novaseq6000进行高通量测序,获得基因表达矩阵;根据文献报道并参考Cellmarker数据库定义细胞亚群;选取EC亚群进行拟时序分析,生成各个细胞分化状态(State)的基因表达矩阵,筛选CNV-EC并初步分析表达特征。另选取6只小鼠建立CNV模型,于造模后7 d取小鼠眼球制备冰冻切片,免疫荧光染色检测EC标志物Pecam1与线粒体外膜蛋白Tomm20、mt-Co1和毛细血管标志物Kdr、Plvap的共定位及面积占比情况,并统计血管直径。结果剪碎复合体和刮取脉络膜所制备单细胞悬液的细胞活率分别为99.4%和99.1%,符合测序要求;流式细胞术检测EC占比约为1.58%。scRNA-seq结果显示,正常对照组和CNV组小鼠脉络膜均包含13个细胞亚群,与正常对照组比较,CNV组视锥/视杆细胞、EC和造血系细胞比例增加,视网膜色素上皮(RPE)细胞和施万细胞比例相应减少。在所有细胞亚群中,EC比例为18.40%。EC拟时序分析显示,脉络膜EC可分为4个State,CNV组中位于State 2状态的EC比例为29.1%,较正常对照组的9.5%明显升高;差异基因分析显示,State 2 EC中线粒体相关基因mt-Nd4和mt-Atp6等表达上调而毛细血管基因Kdr和Esm1等表达显著下调。免疫荧光染色结果显示,CNV区域内Tomm20和mt-Co1在Pecam1阳性EC内�Objective To investigate the molecular expression and pathological features of endothelial cell(EC)in a murine model of choroidal neovascularization(CNV)based on single-cell RNA sequencing(scRNA-seq).Methods Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups,with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid,respectively.Single-cell suspension was prepared by continuous digestion with trypsin/typeⅠcollagenase at 37℃,and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group,with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um(10x Genomics)instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC,revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1,mitochondrial outer membrane proteins Tomm20 and mt-Co1,and capillary markers Kdr and Plvap were observed by immunofluorescence staining,and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University(No.20200181).Results The cell viability of the single-cell suspension prepared from choroid

关 键 词：脉络膜新生血管 内皮细胞 线粒体 毛细血管 单细胞测序 

分 类 号：R773.4[医药卫生—眼科]