单细胞测序对绒山羊毛乳头细胞的鉴定

Identification of Cashmere Dermal Papilla Cells Based on Single-Cell RNA Sequencing Technology

【目的】基于单细胞测序技术探究绒山羊毛乳头细胞标记基因,优化毛乳头细胞体外鉴定的方法,为研究绒山羊毛囊发生发育提供良好的细胞模型。【方法】运用Seurat单细胞分析法分析陕北白绒山羊胚胎期60、90、120 d皮肤组织的单细胞测序数据。原始数据经质控、过滤、标准化后,采用UMAP方法进行数据降维与聚类分析。通过对比已报道的毛囊相关标记基因,获得完整绒山羊毛囊细胞类群谱系信息。通过基因差异表达分析,筛选绒山羊毛乳头细胞特异性标记基因。利用免疫荧光试验鉴定标记蛋白在绒山羊皮肤组织中的表达与分布情况,进一步筛选毛乳头区域特异性表达蛋白。体视镜下机械分离完整绒山羊毛囊,采用酶消化法分离绒山羊毛乳头区域并在体外培养至细胞分离,最终通过差速贴壁法纯化细胞。待毛乳头细胞纯度较高时,利用免疫荧光试验验证候选标记蛋白在分离细胞中的表达情况。【结果】从单细胞水平分析了绒山羊毛囊发生过程中涉及的关键细胞转录信息,成功获得绒山羊皮肤中的17个主要细胞类群信息,鉴定出包括真皮细胞谱系、表皮细胞谱系、毛乳头细胞、毛干细胞、内根鞘细胞等绒山羊皮肤结构关键细胞类群,以及周皮细胞、巨噬细胞、肌肉细胞等其他功能细胞类群。筛选获得毛乳头细胞特异性基因427个,包括SOX2、FGF7、APOD、BMP3、HHIP、HEY2和SPON1等,这些基因在绒山羊毛乳头细胞中的表达丰度远高于其他细胞类型,被认为是毛乳头细胞特异性标记基因。组织免疫荧光试验进一步证明SOX2、FGF7与APOD等蛋白在绒山羊毛乳头区域内特异性表达,可用于皮肤组织中绒山羊毛乳头细胞的定位。此外,本研究在成功分离单根绒山羊次级毛囊的基础上,实现了绒山羊毛乳头区域的贴壁培养,成功观测到大量细胞从毛乳头区域逐步迁移分离的过程。细胞免疫荧光试验结果显示SOX2、FGF7与APOD均在绒山羊毛乳头细胞中表达,且SOX2阳性细胞约占毛乳头细胞总量的76%左右,而FGF7和APOD阳性细胞则占98%以上。结合绒山羊皮肤组织免疫荧光定位结果,SOX2、FGF7与APOD等标记可用于鉴定体外分离培养的绒山羊毛乳头细胞。【结论】利用单细胞测序技术描述了绒山羊主要皮肤细胞的转录组信息,筛选出毛乳头细胞特异性表达基因。经免疫荧光试验证实,单细胞测序鉴定标记基因的方法简单高效,且具备较高准确率。筛选的SOX2、FGF7与APOD不仅为绒山羊毛乳头细胞体内定位提供了有效的标记,而且为多标记鉴定毛乳头细胞提供可能,更为进一步研究毛囊发育相关基因的功能及调控机制奠定重要基础。

【Objective】Based on single-cell RNA sequencing,this article aims to explore the marker genes of cashmere dermal papilla cells,and to optimize the methods to identify dermal papilla cells in vitro,thereby laying a cell model for future pertinent research in cashmere hair follicle development.【Methods】The single-cell transcriptional data from the skin tissues of Shanbei white cashmere embryonic stage(E60,E90 and E120)were analyzed with Seurat package.After quality control,filter and normalization of raw data,the dimension reduction analysis and cell cluster identification were performed by uniform manifold approximation and projection(UMAP).Moreover,depending on cluster-specific expressed gene expression,the principal cell lineage information was identified.The type-specific marker genes of the dermal papilla were obtained after gene expression analysis.The immunofluorescence staining was used to validate the expression position of marker protein to identify the dermal papilla specific protein in goat skin.Whole hair follicles were isolated mechanically under stereoscope,and combined with enzyme detach,cashmere dermal papilla region was isolated and cultured in vitro until cell separation.The dermal papilla cells were purified by different-speed adherence methods.When the cells were highly pure,the expression of candidate marker protein was verified by immunofluorescence assay.【Result】In current study,the key transcription information of goat hair follicle cells was analyzed at single cell level.Information of 17 subsets of cells in cashmere goat skin structure was obtained successfully including dermal cell lineage,epidermal cell lineage,dermal papilla cell,hair stem cell and inner root sheath cell,as well as other functional cell groups such as pericyte cell,macrophage and muscle cell.427 specific markers of dermal papilla cells including SOX2,FGF7,APOD,BMP3,HHIP,HEY2 and SPON1 were screened.By comparison,the expression of these marker genes in cashmere dermal papilla cells was much higher than that in other cell types,which could be confirmed as the specific genes of hair papilla cells.Immunofluorescence result further proved that SOX2,FGF7 and APOD were specifically expressed in the dermal papilla region,and could be used to trace the dermal papilla cells in vivo.In addition,in current study,the single cashmere goat secondary hair follicle was separated successfully,and the adherent culture of dermal papilla was realized.A large number of cells were observed migrating from the hair papilla area.Immunofluorescence assay showed that SOX2,FGF7 and APOD were all expressed in goat dermal papilla cells,and about 76%of cells were SOX2 positive,while more than 98%of cells were FGF7 and APOD positive.Combined with the immunofluorescence results,SOX2,FGF7 and APOD genes factually could be used to identify the cultured goat dermal papilla cells in vitro.【Conclusion】In this study,single cell RNA sequencing technology was used to describe the main transcriptome information of cells in cashmere goat skin,and the specific marker genes of dermal papilla cells were sifted out successfully.And it proved that single-cell sequencing based method was simple and efficient to identify marker genes further identified by immunofluorescence.The discovering of SOX2,FGF7 and APOD not only provided the markers for the localization of hair papilla cells in vivo,but also provided the possibility for the identification of dermal papilla cells with multiple markers,which laid the foundation for further study of the gene functions in regulating hair follicle development.