基于单细胞RNA测序探讨小鼠全层皮肤缺损创面中真皮成纤维细胞的生长因子调控网络

孙礼祥; 吴帅; 张小薇; 刘文杰; 张凌娟

doi:10.3760/cma.j.cn501225-20220215-00029

基于单细胞RNA测序探讨小鼠全层皮肤缺损创面中真皮成纤维细胞的生长因子调控网络

doi: 10.3760/cma.j.cn501225-20220215-00029

厦门大学药学院，细胞应激生物学国家重点实验室，厦门　　361102

基金项目:

国家重点研发计划 2020YFA0112901

国家自然科学基金面上项目 81971551

国家自然科学基金青年科学基金项目 82103702

中国博士后科学基金 2020M682095

详细信息

通讯作者:
张凌娟，Email：lingjuan.zhang@xmu.edu.cn

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出版历程
- 收稿日期: 2022-02-15

Investigation on the growth factor regulatory network of dermal fibroblasts in mouse full-thickness skin defect wounds based on single-cell RNA sequencing

State Key Laboratory of Cell Stress Biology, School of Pharmacy, Xiamen University, Xiamen 361102, China

Funds:

National Key Research and Development Program of China 2020YFA0112901

General Program of National Natural Science Foundation of China 81971551

Youth Science Fund Project of National Natural Science Foundation of China 82103702

China Postdoctoral Science Foundation 2020M682095

More Information

Corresponding author: Zhang Lingjuan, Email: lingjuan.zhang@xmu.edu.cn

摘要

摘要: 目的基于单细胞RNA测序探讨小鼠全层皮肤缺损创面中真皮成纤维细胞（dFb）的异质性与生长因子调控网络。方法采用实验研究方法。取5只8周龄雄性健康C57BL/6小鼠（鼠龄、性别、品系下同）正常皮肤组织，另取5只背部全层皮肤缺损小鼠伤后7 d创面组织，用胶原酶D和DNA酶Ⅰ消化组织获得细胞悬液，用10x Genomics平台构建测序文库，用Illumina Novaseq6000测序仪进行单细胞RNA测序。采用R4.1.1软件的Seurat 3.0程序分析获得2种组织细胞的基因表达矩阵，采用按细胞群、细胞来源、标记皮肤中主要细胞基因分类的二维tSNE云图进行可视化展示。根据已有文献报道和CellMarker数据库检索情况，分析2种组织细胞的基因表达矩阵中标志基因表达情况，对各细胞群进行编号和定义。将基因表达矩阵和细胞分群信息导入R4.1.1软件的CellChat 1.1.3程序，分析2种组织中细胞间通信以及创面组织血管内皮生长因子（VEGF）、血小板衍生生长因子（PDGF）、表皮生长因子（EGF）、成纤维细胞生长因子（FGF）信号通路中的细胞间通信，2种组织中FGF的各亚型与FGF受体（FGFR）各亚型的两两配对（以下简称FGF配受体对）对FGF信号网络的相对贡献，2种组织中相对贡献排名前2的FGF配受体对信号通路中的细胞间通信。取1只健康小鼠正常皮肤组织和1只全层皮肤缺损小鼠伤后7 d创面组织，行多重免疫荧光染色，检测FGF7蛋白的表达与分布及其与二肽基肽酶4（DPP4）、干细胞抗原1（SCA1）、平滑肌肌动蛋白（SMA）和PDGF受体α（PDGFRα）的蛋白共定位表达。结果健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织中均包含25个细胞群，但2种组织中各细胞群中细胞数不一致。PDGFRα、血小板内皮细胞黏附分子1、淋巴管内皮透明质酸受体1、受体型蛋白酪氨酸磷酸酶C、角蛋白10和角蛋白79基因在二维tSNE云图上均有各自明确的分布，分别指示特定的细胞群。将25个细胞群按C0~C24编号，分为9个dFb亚群和16个非dFb群，dFb亚群包括C0间质祖细胞、C5脂肪前体细胞、C13具有收缩能力的肌肉细胞相关成纤维细胞等，非dFb群包括C3中性粒细胞、C8 T细胞、C18红细胞等。与健康小鼠正常皮肤组织比较，全层皮肤缺损小鼠伤后7 d创面组织中细胞间通信更多更密集，其中细胞间通信强度排前3位的细胞群为dFb亚群C0、C1、C2，这3个dFb亚群与创面组织中其他细胞群之间均有通信。在全层皮肤缺损小鼠伤后7 d创面组织中，VEGF信号主要由dFb亚群C0发送、脉管相关细胞群C19和C21接收，PDGF信号主要由周细胞C14发送、多个dFb亚群接收，EGF信号主要由角质形成细胞亚群C9和C11发送、dFb亚群C0接收，FGF信号的主要发送者和接收者均为dFb亚群C6。健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织FGF信号网络中的FGF配受体对相对贡献，均是FGF7-FGFR1排在首位，排名第2的分别是FGF7-FGFR2、FGF10-FGFR1；与正常皮肤组织比较，创面组织FGF7-FGFR1信号通路中的细胞间通信更多，而FGF7-FGFR2和FGF10-FGFR1信号通路中的细胞间通信稍有减少或无明显变化；创面组织FGF7-FGFR1信号通路中的细胞间通信强于FGF7-FGFR2、FGF10-FGFR1信号通路；在2种组织中，FGF7信号均主要由dFb亚群C0与C1和C2发送、dFb亚群C6和C7接收。与健康小鼠正常皮肤组织比较，全层皮肤缺损小鼠伤后7 d创面组织中FGF7蛋白表达更多；在正常皮肤组织中，FGF7蛋白主要表达于皮肤间质层且在靠近真皮白色脂肪组织附近也有表达；在2种组织中，FGF7蛋白与DPP4、SCA1蛋白共定位表达于皮肤间质层中，与PDGFRα蛋白共定位表达于dFb中，与SMA蛋白无共定位表达，其中创面组织中的共定位表达多于正常皮肤组织。结论小鼠全层皮肤缺损创面愈合过程中的dFb存在高异质性，为多种生长因子潜在的主要分泌或接收细胞群落，与生长因子信号通路之间存在紧密、复杂的联系；FGF7-FGFR1信号通路是创面愈合过程中的主要FGF信号通路，靶向调控多个dFb亚群。
- 伤口愈合 /
- 皮肤 /
- 真皮 /
- 成纤维细胞 /
- 成纤维细胞生长因子 /
- 单细胞RNA测序 /
- 生长因子 /
- 细胞间通信
Abstract: Objective To explore the heterogeneity and growth factor regulatory network of dermal fibroblasts (dFbs) in mouse full-thickness skin defect wounds based on single-cell RNA sequencing. Methods The experimental research methods were adopted. The normal skin tissue from 5 healthy 8-week-old male C57BL/6 mice (the same mouse age, sex, and strain below) was harvested, and the wound tissue of another 5 mice with full-thickness skin defect on the back was harvested on post injury day (PID) 7. The cell suspension was obtained by digesting the tissue with collagenase D and DNase Ⅰ, sequencing library was constructed using 10x Genomics platform, and single-cell RNA sequencing was performed by Illumina Novaseq6000 sequencer. The gene expression matrices of cells in the two kinds of tissue were obtained by analysis of Seurat 3.0 program of software R4.1.1, and two-dimensional tSNE plots classified by cell group, cell source, and gene labeling of major cells in skin were used for visual display. According to the existing literature and the CellMarker database searching, the expression of marker genes in the gene expression matrices of cells in the two kinds of tissue was analyzed, and each cell group was numbered and defined. The gene expression matrices and cell clustering information were introduced into CellChat 1.1.3 program of software R4.1.1 to analyze the intercellular communication in the two kinds of tissue and the intercellular communication involving vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) signal pathways in the wound tissue, the relative contribution of each pair of FGF subtypes and FGF receptor (FGFR) subtypes (hereinafter referred to as FGF ligand receptor pairs) to FGF signal network in the two kinds of tissue, and the intercellular communication in the signal pathway of FGF ligand receptor pairs with the top 2 relative contributions in the two kinds of tissue. The normal skin tissue from one healthy mouse was harvested, and the wound tissue of one mouse with full-thickness skin defect on the back was harvested on PID 7. The multiple immunofluorescence staining was performed to detect the expression and distribution of FGF7 protein and its co-localized expression with dipeptidyl peptidase 4 (DPP4), stem cell antigen 1 (SCA1), smooth muscle actin (SMA), and PDGF receptor α (PDGFRα) protein. Results Both the normal skin tissue of healthy mice and the wound tissue of full-thickness skin defected mice on PID 7 contained 25 cell groups, but the numbers of cells in each cell group between the two kinds of tissue were different. Genes PDGFRα, platelet endothelial cell adhesion molecule 1, lymphatic endothelial hyaluronic acid receptor 1, receptor protein tyrosine phosphatase C, keratin 10, and keratin 79 all had distinct distributions on two-dimensional tSNE plots, indicating specific cell groups respectively. The 25 cell groups were numbered by C0-C24 and divided into 9 dFb subgroups and 16 non-dFb groups. dFb subgroups included C0 as interstitial progenitor cells, C5 as adipose precursor cells, and C13 as contractile muscle cells related fibroblasts, etc. Non-dFb group included C3 as neutrophils, C8 as T cells, and C18 as erythrocytes, etc. Compared with that of the normal skin tissue of healthy mice, the intercellular communication in the wound tissue of full-thickness skin defected mice on PID 7 was more and denser, and the top 3 cell groups in intercellular communication intensity were dFb subgroups C0, C1, and C2, of which all communicated with other cell groups in the wound tissue. In the wound tissue of full-thickness skin defected mice on PID 7, VEGF signals were mainly sent by the dFb subgroup C0 and received by vascular related cell groups C19 and C21, PDGF signals were mainly sent by peripheral cells C14 and received by multiple dFb subgroups, EGF signals were mainly sent by keratinocyte subgroups C9 and C11 and received by the dFb subgroup C0, and the main sender and receiver of FGF signals were the dFb subgroup C6. In the relative contribution rank of FGF ligand receptor pairs to FGF signal network in the normal skin tissue of healthy mice and the wound tissue of full-thickness skin defected mice on PID 7, FGF7-FGFR1 was the top 1, and FGF7-FGFR2 or FGF10-FGFR1 was in the second place, respectively; compared with those in the normal skin tissue, there was more intercellular communication in FGF7-FGFR1 signal pathway, while the intercellular communication in FGF7-FGFR2 and FGF10-FGFR1 signal pathways decreased slightly or did not change significantly in the wound tissue; the intercellular communication in FGF7-FGFR1 signal pathway in the wound tissue was stronger than that in FGF7-FGFR2 or FGF10-FGFR1 signal pathway; in the two kinds of tissue, FGF7 signal was mainly sent by dFb subgroups C0, C1, and C2, and received by dFb subgroups C6 and C7. Compared with that in the normal skin tissue of healthy mouse, the expression of FGF7 protein was higher in the wound tissue of full-thickness skin defected mouse on PID 7; in the normal skin tissue, FGF7 protein was mainly expressed in the skin interstitium and also expressed in the white adipose tissue near the dermis layer; in the two kinds of tissue, FGF7 protein was co-localized with DPP4 and SCA1 proteins and expressed in the skin interstitium, co-localized with PDGFRα protein and expressed in dFbs, but was not co-localized with SMA protein, with more co-localized expression of FGF7 in the wound tissue than that in the normal skin tissue. Conclusions In the process of wound healing of mouse full-thickness skin defect wound, dFbs are highly heterogeneous, act as potential major secretory or receiving cell populations of a variety of growth factors, and have a close and complex relationship with the growth factor signal pathways. FGF7-FGFR1 signal pathway is the main FGF signal pathway in the process of wound healing, which targets and regulates multiple dFb subgroups.
- Wound healing /
- Skin /
- Dermis /
- Fibroblasts /
- Fibroblast growth factors /
- Single-cell RNA sequencing /
- Growth factor /
- Intercellular communication
孙礼祥：实验操作、数据整理、论文撰写、经费支持；吴帅：数据处理与论文撰写；张小薇：实验操作；刘文杰：论文修改；张凌娟：研究指导、论文修改、经费支持

所有作者均声明不存在利益冲突

调节性T细胞（Treg）可以通过多种机制来减轻炎症反应和防止自身免疫反应。常驻在外周组织（即非淋巴组织）的Treg有特异性的功能，比如，皮肤组织中的Treg可以促进创面愈合、抑制真皮纤维化、促进表皮再生和增强毛囊生长周期。皮肤组织中的Treg可通过促进影响上皮细胞生物学功能的整合素和TGF-β途径基因的表达，在转录水平上调整组织微环境。研究者观察到，小鼠表皮损伤后皮肤中的Treg促使KC发出急性促炎信号。使用单细胞测序方法，研究者确定了小鼠皮肤损伤时整合素αvβ8会在皮肤Treg上优先表达，Treg可以凭借该整合素激活潜伏的TGF-β，活化的TGF-β则可以直接作用于上皮细胞以促进趋化因子配体5的产生和中性粒细胞的募集。这一信号通路的激活抑制了小鼠表皮的再生，同时降低了金黄色葡萄球菌通过受损的皮肤屏障造成感染的概率。因此，皮肤中表达αvβ8的Treg与其典型的免疫抑制功能似乎存在矛盾，在皮肤屏障的完整性丧失后表现为促进炎症的急性发作，进而促进宿主对细菌感染的防御。

张凡，编译自《Sci Immunol》, 2021,6(62):eabg2329；肖健，审校

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1 基于健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织细胞单细胞RNA测序数据的、按细胞群分类与细胞来源分类的二维tSNE云图。1A.细胞群分类；1B.细胞来源分类

注：图1A中不同细胞群用不同颜色展示，并用0~24编号；图1B中橙色标记的细胞来自正常皮肤组织，蓝色标记的细胞来自创面组织

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2 基于健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织细胞单细胞RNA测序数据的、标记皮肤中主要细胞的基因在细胞群中分布的二维tSNE云图。2A.血小板衍生生长因子受体α基因；2B.血小板内皮细胞黏附分子1基因+淋巴管内皮透明质酸受体1基因；2C.受体型蛋白酪氨酸磷酸酶C基因；2D.角蛋白10基因+角蛋白79基因

注：图例表示基因表达强度

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3 基于健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织细胞单细胞RNA测序数据的、表达血小板衍生生长因子受体α基因的、真皮成纤维细胞亚群的特定基因表达情况气泡图

注：从左至右各列分别对应细胞群C0、C5、C7、C1、C2、C4、C17、C6、C13；左侧缩写与中文均为基因名称，DPP4为二肽基肽酶4，LY6A为淋巴细胞抗原6A，ICAM1为细胞间黏附分子1，CRAMP为组织蛋白酶抑制素，PLIN2为脂滴包被蛋白2，CXCL为C-X-C基序趋化因子，IL-4为白细胞介素4，VCAM1为血管细胞黏附分子1，PCNA为增殖细胞核抗原，SAA3为血清淀粉样蛋白A-3，LGR5为富含亮氨酸重复的G蛋白偶联受体5，CSF1R为集落刺激因子1受体，COLⅠα1为Ⅰ型胶原α1链蛋白，ENTPD1为外核苷三磷酸二磷酸水解酶1，LRIG1为亮氨酸丰富重复免疫球蛋白样域蛋白1，LEF1为淋巴增强子结合因子1，ALPL为肝肾骨碱性磷酸酶，ACTα2为肌动蛋白α2，TAGLN为肌动蛋白凝胶蛋白，MYL1为肌球蛋白轻链1，MYLPF为肌球蛋白可磷酸化调节轻链

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4 基于健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织细胞单细胞RNA测序数据的、非真皮成纤维细胞群的特定基因表达情况热图

注：从左至右各列分别对应细胞群C3、C10、C12、C15、C22、C8、C20、C18、C9、C11、C14、C16、C19、C21、C23、C24；左侧缩写与中文均为基因名称，PTPRC为受体型蛋白酪氨酸磷酸酶C，LY6A为淋巴细胞抗原6A，S100A8为S100钙结合蛋白A8，MRC1为甘露糖受体C1，CMA1为糜蛋白酶1，TPSβ2为类胰蛋白酶β2，NKG为自然杀伤细胞相关蛋白，GYPA为血型糖蛋白A，CSPG4为硫酸软骨素蛋白聚糖4，PDGFRβ为血小板衍生生长因子受体β，RGS5为G蛋白信号通路调节蛋白5，NGFR为神经生长因子受体，GFRα3为胶质细胞源性神经营养因子家族受体α3，UGT8A为尿苷二磷酸糖基转移酶8A，TIE1为免疫球蛋白样表皮生长因子样域酪氨酸激酶1，PECAM1为血小板内皮细胞黏附分子1，LYVE1为淋巴管内皮透明质酸受体1，TYRP1为5,6-二羟基吲哚-2-羧酸氧化酶，DCT为多巴色素互变异构酶，ACTα2为肌动蛋白α2，TAGLN为肌动蛋白凝胶蛋白，MYH11为肌球蛋白轻链11

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5 基于健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织细胞单细胞RNA测序数据的细胞间通信网络图与热图。5A、5B.分别为正常皮肤组织、创面组织中细胞间通信情况网络图，图5B中细胞间通信较图5A更多更密集；5C.创面组织中细胞间通信强度热图；5D、5E、5F.分别为创面组织中细胞群C0、C1、C2与其他细胞群通信情况网络图

注：图5A、5B、5D、5E、5F中从最右侧红点开始，逆时针排列的25个点代表25个细胞群，对应编号为C0~C24，图中连线代表细胞间通信，连线的颜色与信号发送细胞群颜色一致，连线的粗细代表信号强度，点大小代表细胞群中细胞量；图5C中从左侧顶部/底部最左侧红色块开始，向下/右排列的25个色块代表25个细胞群，对应编号为C0~C24

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6 基于全层皮肤缺损小鼠伤后7 d创面组织细胞单细胞RNA测序数据的特定生长因子信号通路中细胞间通信热图。6A.血管内皮生长因子信号通路；6B.血小板衍生生长因子信号通路；6C.表皮生长因子信号通路；6D.成纤维细胞生长因子信号通路

注：色块颜色越接近深绿色代表信号强度越强，色块颜色越接近白色代表信号强度越弱；各图中从底部最左侧红色块开始，向右排列的25个色块代表25个细胞群，对应编号为C0~C24

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7 基于健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织细胞单细胞RNA测序数据的、成纤维细胞生长因子（FGF）的各亚型与FGF受体（FGFR）各亚型的两两配对在FGF信号网络中贡献的相对贡献图。7A.正常皮肤组织；7B.创面组织

注：以排名第1的配受体对为标准，其他配受体对相对贡献以其与排名第1的配受体对相对贡献的比例表示

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8 基于健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织细胞单细胞RNA测序数据的、成纤维细胞生长因子（FGF）的各亚型与FGF受体（FGFR）各亚型的两两配对信号通路中的细胞间通信网络图。8A、8B、8C.分别为正常皮肤组织中FGF7-FGFR1、FGF7-FGFR2、FGF10-FGFR1信号通路中的细胞间通信，图8A中的细胞间通信较少，图8B中的细胞间通信较多，图8C中的细胞间通信少；8D、8E、8F.分别为创面组织中FGF7-FGFR1、FGF7-FGFR2、FGF10-FGFR1信号通路中的细胞间通信，图8D中细胞间通信明显多于图8A，图8E中细胞间通信稍少于图8B，图8F中细胞间通信与图8C相近，图8E、8F中细胞间通信明显少于图8D

注：网络图中从最右侧红点开始，逆时针排列的25个点代表25个细胞群，对应编号为C0~C24；网络图中连线代表细胞间通信，连线的颜色与信号发送细胞群颜色一致，连线的粗细代表信号强度，点大小代表细胞群中细胞量

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9 健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后7 d创面组织中FGF7蛋白表达与分布及其与DPP4、SCA1、SMA、PDGFRα蛋白的共定位表达花青素3-异硫氰酸荧光素-花青素5-4',6-二脒基-2-苯基吲哚×200，图中标尺为2 mm。9A、9B、9C、9D与9E、9F、9G、9H.分别为正常皮肤组织与创面组织中FGF7染色、DPP4染色、SCA1染色、FGF7与DPP4和SCA1及细胞核染色的重叠图片，图9A中FGF7蛋白表达量少，主要位于皮肤间质层且在靠近真皮白色脂肪组织附近也有表达，图9B中DPP4蛋白表达较多，图9C中SCA1蛋白表达较多，图9D中FGF7与DPP4和SCA1的蛋白共定位表达较少，图9E中FGF7蛋白表达量明显多于图9A，图9F中DPP4蛋白表达量与图9B相近，图9G中SCA1蛋白表达量与图9C相近，图9H中FGF7与DPP4和SCA1的蛋白共定位表达明显多于图9D；9I、9J、9K、9L与9M、9N、9O、9P.分别为正常皮肤组织与创面组织中FGF7染色、SMA染色、PDGFRα染色、FGF7与SMA和PDGFRα及细胞核染色的重叠图片，图9I中FGF7蛋白表达部位与图9A相近但量较少，图9J中SMA蛋白表达量少，图9K中PDGFRα蛋白表达量较少，图9L中FGF7蛋白与SMA蛋白无共定位且和PDGFRα蛋白的共定位表达少，图9M中FGF7蛋白表达量明显多于图9I，图9N中SMA蛋白表达明显多于图9J，图9O中PDGFRα蛋白表达与图9K相近，图9P中FGF7蛋白与SMA蛋白无共定位且与PDGFRα蛋白的共定位表达明显多于图9L

注：成纤维细胞生长因子7（FGF7）阳性染色为红色，二肽基肽酶4（DPP4）与平滑肌肌动蛋白（SMA）阳性染色均为绿色，干细胞抗原1 （SCA1）与血小板衍生生长因子受体α（PDGFRα）阳性染色均为蓝紫色，细胞核染色为白色；绿色箭头指示FGF7蛋白在间质层的分布，黄色箭头指示FGF7蛋白在白色脂肪组织附近的分布，红色箭头指示FGF7蛋白与其他蛋白共定位位置

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