慢性皮肤溃疡的细胞异质性的单细胞转录组测序及临床意义分析

Single-cell transcriptome sequencing and clinical significance analysis of cellular heterogeneity in chronic skin ulcers

摘要:

背景与目的 目前，慢性皮肤溃疡仍是影响患者日常生活及心理负担的重要疾病，其组织内细胞及细胞外基质的异常可能破坏微环境内的平衡，从而阻碍正常的皮肤修复进程，导致溃疡迁延不愈。其发生发展机制及诊断标志物尚无相关研究。单细胞测序是近年来新发展的高通量测序方法，通过对单细胞分辨率下的基因测序，精准地揭示疾病的机制，在多种疾病中均有研究。本研究利用单细胞转录组测序（scRNA-Seq）技术检测慢性皮肤溃疡组织细胞异质性，以期阐明慢性溃疡难以愈合的潜在分子机制，并为临床治疗提供新思路。方法 通过scRNA-Seq技术，比较慢性溃疡组织和正常皮肤组织在细胞亚群及基因表达层面的差异。使用微流控平台对单细胞进行分选，并构建cDNA文库，随后进行差异基因分析和功能富集分析。结果 scRNA-Seq分析揭示了慢性溃疡组织的显著免疫－代谢重塑特征：溃疡组织中B细胞、单核细胞及巨噬细胞数量较对照正常组织增加2.1~3.5倍，同时伴随胶原合成基因（COL1A1/COL3A1）的广泛激活与免疫调节因子（如颗粒酶家族GZMA/GZMB/H）的协同抑制。跨细胞亚群的功能网络分析显示，HIF-1信号介导的缺氧应答与PI3K/Akt通路异常形成正反馈环路，加剧炎症因子（CXCL3/8，TGFBI）的分泌失衡及线粒体氧化磷酸化代偿性上调。结论 慢性皮肤溃疡在细胞异质性和基因表达方面有显著差异，提示慢性溃疡不仅是单纯的组织缺损，更是由长期炎症和免疫失调主导的复杂病理过程。慢性溃疡微环境中多细胞亚群的协同失调，其持续性炎症反应与代谢异常通过HIF-1/TNF/MAPK通路网络相互关联。颗粒酶基因家族下调及组蛋白修饰异常可能共同导致免疫清除缺陷，为开发靶向表观调控或线粒体功能的新型疗法提供理论依据。

Abstract:

Background and Aims Chronic skin ulcers are a significant disease affecting patients' daily lives and psychological well-being. Abnormalities in the cells and extracellular matrix within the tissue may disrupt the balance of the microenvironment, hindering the normal skin repair process and leading to delayed healing of the ulcer. There is currently a lack of research on the mechanisms underlying the development of chronic ulcers and their diagnostic biomarkers. Single-cell sequencing, a newly developed high-throughput sequencing method in recent years, uses gene sequencing at the single-cell resolution to precisely reveal disease mechanisms and has been applied in various diseases. This study used single-cell transcriptome sequencing (scRNA-Seq) to investigate the cellular heterogeneity in chronic skin ulcer tissue to elucidate the potential molecular mechanisms behind delayed healing and provide new insights for clinical treatment.Methods The scRNA-Seq technology was used to compare the differences in cell subpopulations and gene expression between chronic ulcer tissue and normal skin tissue. Single cells were sorted using a microfluidic platform, and cDNA libraries were constructed for subsequent differential gene analysis and functional enrichment analysis.Results scRNA-Seq analysis revealed significant immune-metabolic remodeling features in chronic ulcer tissue: the number of B cells, monocytes, and macrophages in ulcer tissue increased by 2.1 to 3.5 times compared to the normal tissue control. This was accompanied by widespread activation of collagen synthesis genes (COL1A1/COL3A1) and synergistic suppression of immune regulators (e.g., granzyme family GZMA/GZMB/H). Cross-cell subpopulation functional network analysis showed that hypoxia response mediated by the HIF-1 signaling pathway and PI3K/Akt pathway abnormalities formed a positive feedback loop, exacerbating the imbalance in the secretion of inflammatory factors (CXCL3/8, TGFBI) and compensatory upregulation of mitochondrial oxidative phosphorylation.Conclusion Chronic skin ulcers exhibit significant differences in cellular heterogeneity and gene expression, suggesting that chronic ulcers are not simply tissue defects but a complex pathological process dominated by chronic inflammation and immune dysregulation. The coordinated dysregulation of multiple cell subpopulations in the ulcer microenvironment, along with persistent inflammatory responses and metabolic abnormalities, is interconnected through the HIF-1/TNF/MAPK pathway network. Downregulation of granzyme gene family members and abnormal histone modifications may contribute to immune clearance defects, providing a theoretical basis for developing novel therapies targeting epigenetic regulation or mitochondrial function.