基于单细胞测序技术构建小鼠磨牙牙髓细胞的发育图谱  

Single-cell transcriptome analysis reveals development atlas of mouse molar pulp cells

作　　者：温泉 任惠惠 赵玉鸣[2] 闫文娟 葛立宏[2] 陈小贤[1] Wen Quan;Ren Huihui;Zhao Yuming;Yan Wenjuan;Ge Lihong;Chen Xiaoxian(First Clinical Division,Peking University School and Hospital of Stomatology&National Center for Stomatology&National Clinical Research Center for Oral Diseases&National Engineering Research Center of Oral Biomaterials and Digital Medical Devices&Beijing Key Laboratory of Digital Stomatology,Beijing 100034,China;Department of Pediatric Dentistry,Peking University School and Hospital of Stomatology&National Center for Stomatology&National Clinical Research Center for Oral Diseases&National Engineering Research Center of Oral Biomaterials and Digital Medical Devices&Beijing Key Laboratory of Digital Stomatology,Beijing 100081,China)

机构地区：[1]北京大学口腔医学院·口腔医院第一门诊部、国家口腔医学中心、国家口腔疾病临床医学研究中心、口腔生物材料和数字诊疗装备国家工程研究中心、口腔数字医学北京市重点实验室,北京100034 [2]北京大学口腔医学院·口腔医院儿童口腔科、国家口腔医学中心、国家口腔疾病临床医学研究中心、口腔生物材料和数字诊疗装备国家工程研究中心、口腔数字医学北京市重点实验室,北京100081

出　　处：《中华口腔医学杂志》2023年第5期442-450,共9页Chinese Journal of Stomatology

基　　金：北京市自然科学基金(7212135,L222144);北京大学口腔医学院青年科研基金(PKUSS20220113)。

摘　　要：目的通过在单细胞水平分析小鼠磨牙细胞间异质性,构建牙髓细胞发育的时空图谱,进一步揭示牙齿发育的过程和调控机制。方法分别收集出生后0、3 d的C57BL/6小鼠下颌第一磨牙各10颗,制备单细胞悬液,采用单细胞转录组测序(single-cell RNA sequencing,scRNA-seq)技术进行测序。从本课题组前期研究中提取出生后7 d小鼠磨牙scRNA-seq数据,与0、3 d数据进行合并分析。使用Seurat程序对测序数据进行质控、标准化、降维和聚类分析;Monocle程序进行拟时序分析,预测发育轨迹;Scillus程序进行基因本体分析,对细胞功能进行注释。使用原位杂交技术对标记基因进行体内定位,明确不同亚群细胞的体内分布和时空变化。结果Seurat分析显示小鼠磨牙细胞具有26个细胞亚群,可分为牙髓细胞、牙囊细胞、上皮细胞、免疫细胞、内皮细胞、管周细胞、胶质细胞和红细胞等八大类细胞,其中牙髓细胞包含5个亚群:成熟牙髓细胞,标记基因为柯萨奇病毒腺病毒受体(coxsackie virus and adenovirus receptor,Cxadr);牙乳头细胞,标记基因为SPARC相关模块化钙结合蛋白2(SPARC related modular calcium binding 2,Smoc2);周期细胞,标记基因为Ⅱ型DNA拓扑异构酶(DNA topoisomeraseⅡalpha,Top2a);前成牙本质细胞,标记基因为棕榈酰蛋白羧酸酯酶(notum palmitoleoyl-protein carboxylesterase,Notum);成牙本质细胞,标记基因为牙本质涎磷蛋白(dentin sialophosphoprotein,Dspp)。随着牙胚发育,成熟牙髓细胞的比例逐渐增加,周期细胞和成牙本质细胞比例逐渐降低。原位杂交染色和基因本体分析结果显示,Cxadr+成熟牙髓细胞定位于牙冠上部,主要功能为成骨和“细胞外结构组织”;Smoc2+牙乳头细胞位于根尖,主要功能与“细胞外结构组织”和器官发育相关;Top2a+周期细胞位于牙冠下部,进行有丝分裂;Notum+前成牙本质细胞邻近上皮根鞘,和Dspp+成牙本质细胞的主要功能Objective Single-cell RNA sequencing(scRNA-seq)was used to analyze the developing mouse molars,in order to construct a spatiotemporal development atlas of pulp cells,and further to reveal the developmental process and regulatory mechanism of tooth development.Methods Ten mandibular first molars from C57BL/6 mice in postnatal day(PN)0 and 3 were respectively dissected and digested to obtain single-cell suspensions.scRNA-seq was performed on 10×Genomics platform.PN 7 mouse molar scRNA-seq data were obtained from our previous study.PN 0,3,and 7 scRNA-seq data were integrated for following analysis.The initial quality control,mapping and single cell expression matrix construction were performed by Cell Ranger.Quality control,standardization,dimensional reduction and cluster analysis were performed by using Seurat.Monocle was used to generate the pseudotime trajectory.Scillus was used to perform gene ontology analysis.In order to detect the spatiotemporal change of different population of pulp cells,the marker genes of each cluster were demonstrated by RNAscope in situ hybridization.Results There were twenty-six cell clusters within mouse molars,which were identified as eight different cell types,including dental pulp cells,dental follicle cells,epithelial cells,immune cells,endothelial cells,perivascular cells,glial cells and erythrocytes.We further re-clustered and analyzed dental pulp cells.Cluster 0 were mature pulp cells,which located at the upper portion of crown.The main functions of cluster 0 were osteogenesis and extracellular structure organization.Cluster 1 were apical papilla cells,which located at the apical part of roots,whose main functions were extracellular structure organization and organ development.Cluster 2 were cycling cells,which were actively proliferated,resided in the lower portion of the crown.Cluster 3 and 4 were preodontoblasts and odontoblasts,respectively.Their functions were closely related to biomineralization.The proportion of mature pulp cells increased with the development process,

关 键 词：牙髓 牙乳头 单细胞转录组测序 牙齿发育 成牙本质细胞 细胞图谱 小鼠 近交C57BL 

分 类 号：R781[医药卫生—口腔医学]