P227 Molecular and functional characterisation of distinct resident and migratory skin fibroblast populations in systemic sclerosis
P227 Molecular and functional characterisation of distinct resident and migratory skin fibroblast populations in systemic sclerosis
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P227 系统性硬化症中不同驻留和迁移皮肤成纤维细胞群的分子和功能特征
DOI：
10.1093/rheumatology/keac133.226
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发表时间：
2022
期刊：
影响因子：
5.5
通讯作者：
Clark K
中科院分区：
文献类型：
--
作者：
Clark K
背景/目标现代单细胞分析已证实正常和患病皮肤内成纤维细胞群体的功能异质性。为了更好地了解成纤维细胞亚群之间的差异，我们开发了一种新技术，可以从健康对照（HC）和系统性硬化症（SSc）皮肤中选择性分离“迁移”成纤维细胞和非迁移“常驻”成纤维细胞，并使用纤维化潜力和基因表达的功能测定来比较这些新型成纤维细胞群体。 从 dcSSc (n = 3) 和 HC 皮肤 (n = 3) 收集。通过标准外植体培养分离迁移成纤维细胞。然后对剩余的活检碎片进行胶原酶消化，产生常驻成纤维细胞群。这些群体在第 3-6 代得到扩展和使用。功能表征包括 3-D 胶原凝胶收缩和迁移性划伤试验。通过蛋白质印迹比较前胶原蛋白 I、CTGF 和 alphaSMA 的表达。对每个成纤维细胞群体进行批量 RNAseq。使用R软件“tidyverse”进行统计分析。基因表达的显着差异是倍数变化≥ 1.5，调整后的 p 值 (FDR) < 0.05。结果与 HC 相比，SSc 成纤维细胞表现出标志性纤维化表型，包括凝胶收缩增加、迁移更快以及 Col1、CTGF 和 alphaSMA 过度表达。相反，与 SSc 和 HC 中的迁移性成纤维细胞相比，常驻成纤维细胞表现出收缩、迁移减少和 αSMA 分泌减少。 RNAseq证实了SSc驻留和迁移成纤维细胞之间有1483个显着差异表达的基因，而HC驻留和迁移成纤维细胞之间没有显着差异表达的基因。为了进一步了解两个成纤维细胞群体之间与疾病相关的差异，我们比较了 SSc 和 HC 迁移成纤维细胞以及常驻成纤维细胞之间的基因表达。与 HC 相比，SSc 的迁移成纤维细胞中有 644 个基因上调（包括 ACTA2、C1QTNF3）。与 HC 相比，SSc 驻留成纤维细胞中有 914 个基因上调（包括 IL6、CCL2）。与 HC 相比，这 375 个基因在 SSc 成纤维细胞群体中均上调（表 1），包括 IL11 和 COMP.P227 表 1 成纤维细胞群体和 HC 之间前 10 个显着差异表达的基因 仅显着上调 SSc 迁移成纤维细胞与 HC 相比，在 SSc 迁移成纤维细胞和常驻成纤维细胞中均显着上调，与 HC 相比 仅显着上调 SSc 驻留成纤维细胞对比 HC Log2FC SSc 对比 HC Log2FC 迁移 SSc 对比 HC Log2FC 驻留 SSc 对比 HC Log2FC SSc 对比 HC ID1 2.824 5.306 KANK4 5.659 HBEGF 6.229 TGFB2 2.577 4.941 ST6GAL2 4.168 EGR2 4.712麦克姆2.319 2.458 CAVIN4 4.391 PTGS2 3.192 ACTA2 2.016 4.007 AMIGO2 3.281 AQP1 2.779 PLCB4 1.937 3.886 TSPAN2 4.597 …
Background/AimsModern single-cell analysis has confirmed functional heterogeneity amongst fibroblast populations within normal and diseased skin. To better understand differences between fibroblast subpopulations, we have developed a novel technique to selectively isolate “migratory” fibroblasts, and non-migratory “resident” fibroblasts from heathy control (HC) and systemic sclerosis (SSc) skin and used functional assays of fibrotic potential, and gene expression to compare these novel fibroblasts populations.MethodsForearm skin punch biopsies were collected from dcSSc (n = 3), and HC skin (n = 3). Migratory fibroblasts were isolated by standard explant culture. The remaining biopsy fragments then underwent collagenase digestion to yield a population of resident fibroblasts. These populations were expanded and used at passage 3-6. Functional characterisation included 3-D collagen gel contraction, and migratory scratch-wound assays. Expression of pro-collagen I, CTGF and alphaSMA was compared by western blot. Bulk RNAseq on each fibroblast population was performed. Statistical analysis was carried out using Rsoftware “tidyverse”. Significant differences in gene expression were those with a fold change of ≥ 1.5, and adjusted p-value (FDR) of < 0.05.ResultsSSc fibroblasts showed a hallmark fibrotic phenotype with increased gel contraction, faster migration, and overexpression of Col1, CTGF and alphaSMA compared with HC. Conversely, resident fibroblasts showed reduced contraction, migration, and reduced secretion of alphaSMA compared to migratory fibroblasts in both SSc and HCs. RNAseq confirmed 1483 significantly differentially expressed genes between SSc resident and migratory fibroblasts, whereas no genes were significantly differentially expressed between HC resident and migratory fibroblasts. To further understand disease-related differences between the two fibroblast populations, we compared gene expression between SSc and HC migratory fibroblasts, and resident fibroblasts. 644 genes were upregulated in the migratory fibroblasts of SSc compared to HC (including ACTA2, C1QTNF3). 914 genes were upregulated in SSc resident fibroblasts compared to HC (including IL6, CCL2). Of these 375 genes were upregulated in both SSc fibroblast populations compared to HC (Table 1) including IL11 and COMP.P227Table1top 10 significantly differentially expressed genes between fibroblast populations and HCSignificantly upregulated only SSc migratory fibroblasts vs HC Significantly upregulated in both SSc migratory and resident fibroblasts vs HC Significantly upregulated only SSc resident fibroblasts vs HC Log2FC SSc vs HC Log2FC migratory SSc vs HC Log2FC resident SSc vs HC Log2FC SSc vs HC ID1 2.824 5.306 KANK4 5.659 HBEGF 6.229 TGFB2 2.577 4.941 ST6GAL2 4.168 EGR2 4.712 MCAM 2.319 2.458 CAVIN4 4.391 PTGS2 3.192 ACTA2 2.016 4.007 AMIGO2 3.281 AQP1 2.779 PLCB4 1.937 3.886 TSPAN2 4.597 …