关键词: 单细胞测序, 绒山羊, 毛乳头细胞, 标记基因, 鉴定

Abstract:

【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.

Key words: single-cell RNA sequencing, cashmere, dermal papilla cells, marker gene, identification