天祝白牦牛退行期毛囊细胞亚群鉴定以及特征基因生物信息学分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1556

摘要/Abstract

摘要：

从单细胞水平解析天祝白牦牛退行期毛囊发育过程主要细胞类型，旨在对主要细胞类群特异表达基因进行生物功能预测与生物信息学分析，探索退行期毛囊发育调控机制。采用单细胞转录组测序对退行期毛囊进行异质性分析，利用已知标记分子对细胞类群进行筛选鉴定，并对鉴定得到的主要细胞类群进行GO和KEGG分析，同时对毛囊形态发生过程中涉及的关键分子进行免疫组织分析。结果表明，天祝白牦牛退行期涉及IFE-DC细胞、表皮细胞系、黑色素细胞、INFU细胞等细胞类群。其特征基因主要参与表皮发育、上皮细胞分化、超纤维组织发育、组织形态发生以及细胞形态学发生等生物过程。KEGG富集分析发现，特征基因主要富集在黏附连接、细胞周期、RNA转运等与毛囊发育相关的通路中。涉及的不同细胞类型拥有GJA1、FKBP4、KRT1、KRT80、FGFR2等28个共同基因。该研究成功鉴定出天祝白牦牛退行期主要细胞类群，获得了特征基因富集通路，揭示了退行期毛囊发育机制。

关键词: 天祝白牦牛, 退行期, 毛囊, 单细胞转录组测序, 生物信息学分析

Abstract:

The main cell types in the hair follicle development process of Tianzhu white yak during the catagen were analyzed at the single cell level，aiming to perform functional prediction and bioinformatics analysis on the characteristic genes of the main cell groups，and to explore the hair follicle development mechanism during catagen. Single cell RNA sequencing was used to analyze the heterogeneity of degenerative hair follicles during catagen，and known marker molecules were used to screen and identify cell groups. GO and KEGG analysis were performed on the main identified cell groups，and immune tissue analysis was performed on the key molecules involved in hair follicle morphogenesis. The results showed that catagen of Tianzhu white yak was involved in IFE-DC cells，epidermal cell lines，melanocytes，INFU and other cell groups. Its characteristic genes were mainly involved in biological processes such as epidermis development，epithelial cell differentiation，super fiber tissue development，tissue morphogenesis and cell morphology. KEGG enrichment analysis discovered that the characteristic genes were mainly enriched in adhesion connection，cell cycle，RNA transport and other pathways related to hair follicle development. The different involved cell types contained 28 common genes such as GJA1，FKBP4，KRT1，KRT80 and FGFR2. This study successfully identified the main cell groups of Tianzhu white yak during catagen，obtained the enrichment pathways of characteristic genes，and revealed the hair follicle development mechanism during catagen.

Key words: Tianzhu white yak, catagen, hair follicle, single cell RNA sequencing, bioinformatics analysis

郑青波, 叶娜, 张哓兰, 包鹏甲, 王福彬, 任稳稳, 廖月姣, 阎萍, 潘和平. 天祝白牦牛退行期毛囊细胞亚群鉴定以及特征基因生物信息学分析[J]. 生物技术通报, 2022, 38(10): 262-272.

ZHENG Qing-bo, YE Na, ZHANG Xiao-lan, BAO Peng-jia, WANG Fu-bin, REN Wen-wen, LIAO Yue-jiao, YAN Ping, PAN He-ping. Identification of Hair Follicle Cell Subsets and Bioinformatics Analysis of Characteristic Genes in Tianzhu White Yak During Catagen[J]. Biotechnology Bulletin, 2022, 38(10): 262-272.

图/表 12

表1 细胞类型标记分子

Table 1 Cell type marker molecules

亚群Cluster ID	标记基因 Marker gene	类型Cell type	参考文献Reference
0，3，8，9	KRT14，KRT15，KRT5	表皮细胞Epidermal	[5]
1，4，10	KRT1，KRT10，KRTDAP，SBSN	滤泡间上皮分化细胞 Interfollicular epidermis differentiated cell	[5]
2，6	SPARC，LHX2	毛干细胞 Hair shaft	[6]
5	KRT28，KRT27，KRT71	内根鞘细胞 Inner root sheath	[7]
7	TOP2A，UBE2C	漏斗细胞 Infundibulum	[5]
13	PMEL，TYRP1	黑素细胞Melanocyte	[7-8]

图1 天祝白牦牛皮肤组织结构图 ORS：外根鞘；IRS：内根鞘；PF：初级毛囊；SF：次级毛囊；HS：毛干

Fig. 1 Skin tissue structure of Tianzhu white yak ORS：Outer root sheath. IRS：Inner root sheath. PF：Primary follicles. SF：Secondary follicles. HS：Hair shaft

图2 检测到的细胞中基因数与UMI总数统计以及相关性分析 A：细胞基因数与UMI总数统计；B：基因数与UMI总数相关性分析。每个点表示一个细胞，nFeature_RNA 纵坐标表示每个细胞中表达的基因数，nCount_RNA 纵坐标表示每个细胞中总 UMI 数，percent.mt 纵坐标表示每个细胞中线粒体基因的 UMI 占该细胞总 UMI 的比例，横坐标则表示细胞在每个纵坐标值上的丰度

Fig. 2 Statistics and correlation analysis of gene number and total UMI in detected cells A：Number of cell genes and total UMI statistics. B：Correlation analysis between gene number and total UMI. Each point represents a cell，the nFeature _ RNA ordinate represents the number of genes expressed in each cell，nCount _ RNA ordinate represents the total UMI number in each cell，percent.mt ordinate represents the ratio of mitochondrial gene UMI to total UMI in each cell，and the abscissa indicates the abundance of cells in each ordinate value

图3 基因离散度分布图

Fig. 3 Gene dispersion distribution map

图4 主成分分析 A：PCA降维；B：部分基因PCA分析；C：确定数据集分群个数

Fig. 4 Principal component analysis A：PCA dimension reduction. B：Partial gene PCA analysis. C：Determine the number of data set clusters

图5 UMAP聚类与主要类型细胞鉴定 A：UMAP聚类结果中细胞分群信息；B：UMAP聚类结果中主要细胞类型鉴定；C：特异性标记基因在不同细胞中的表达水平；D：特异性标记分子在部分cluster中的表达情况

Fig.5 UMAP clustering and identification of major cell types A：Cell clustering information in the UMAP plot. B：Identification of major cell types in UMAP plot. C：The expressions of specific marker genes in different cells. D：Expression of specific markers in some clusters

图6 不同细胞类群差异基因比较分析

Fig.6 Comparative analysis of differential genes in different cell clusters

图7 表皮细胞系与IFE-DC细胞不同cluster特征基因富集分析 A：表皮细胞谱系不同cluster特征基因富集分析；B：IFE-DC细胞不同cluster特征基因富集分析

Fig. 7 Enrichment analysis of different clusters characteristic genes in epidermal cell lineage and IFE-DC A：Enrichment analysis of different clusters characteristic genes in epidermal cell lineage. B：Enrichment analysis of different clusters characteristic genes in IFE-DC

图8 HS与黑色素细胞特征基因富集分析 A：HS特征基因富集分析；B：黑色素细胞特征基因富集分析

Fig.8 Enrichment analysis of HS and melanocytes characteristic genes A：Enrichment analysis of HS characteristic genes. B：Enrichment analysis of melanocyte characteristic genes

图9 INFU细胞与IRS细胞特征基因的GO富集分析 A：INFU细胞特征基因的GO富集分析；B：IRS细胞特征基因的GO富集分析

Fig.9 GO enrichment analysis of characteristic genes in INFU and IRS cells A：GO enrichment analysis of INFU characteristic genes. B：GO enrichment analysis of IRS characteristic genes

图10 不同细胞类群特征基因KEGG富集分析 A：不同细胞KEGG富集图；B：特征基因互作网络图

Fig. 10 KEGG Enrichment analysis of characteristic gene in different cell clusters A：KEGG enrichment map of different cells. B：Characteristic gene interaction network diagram

图11 关键标记基因荧光免疫组化分析

Fig.11 Fluorescence immunohistochemical analysis of key marker genes

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