[摘 要] 目的：通过多组学整合分析，解析肠道菌群在胆管癌（CCA）发生发展中的潜在作用机制并识别相关关键基因。方法：基于SRA数据库的16S rRNA测序数据，比较CCA患者与健康对照者的肠道菌群组成；采用孟德尔随机化（MR）分析评估菌群与CCA风险的遗传关联。通过gutMGene和GeneCards数据库获取相关代谢物与基因，进行代谢和功能富集分析。整合GEO单细胞转录组数据（GSE213452），解析肿瘤微环境的细胞组成，重点关注T细胞亚群及其功能状态，并结合TCGA-CHOL数据集验证关键候选基因的表达差异。结果：与健康对照组相比，CCA患者肠道菌群组成发生显著改变，变形菌门下菌群异常富集（LDA > 4）。MR分析进一步证实，肠杆菌目与肠杆菌科的遗传易感性均与CCA风险呈正向关联。代谢通路富集分析提示，菌群相关代谢物主要参与嘌呤代谢及糖酵解/糖异生等通路；功能富集分析显示，相关基因显著富集于NOD样受体、IL-17、Toll样受体及NF-κB信号通路等炎症免疫通路。单细胞转录组分析结果显示，CCA组织中肿瘤细胞比例显著升高（P < 0.05），T细胞比例由20.7%增至39.2%；拟时序分析结果表明，MKI67+ T细胞处于分化末期并呈高增殖特征，其差异基因与菌群相关基因存在交集，其中SERPINA1和IFNG表达在肿瘤免疫微环境中显著变化（P < 0.001），可能发挥核心调控作用。TCGA-CHOL数据集验证显示，SERPINA1在CCA肿瘤组织中显著下调（P < 0.001），而IFNG在肿瘤与正常组织间无显著差异（P > 0.05）。结论：肠道菌群失衡（尤其是肠杆菌科异常增殖）可能通过代谢-免疫调控网络促进CCA进展，T细胞功能变化与关键基因（SERPINA1和IFNG）在MKI67+ T细胞中的差异化表达模式密切相关。

[Abstract] Objective: To comprehensively elucidate the potential mechanisms of gut microbiota in the occurrence and development of cholangiocarcinoma (CCA) and identify related key genes through integrative multi-omics analysis. Methods: Based on 16S rRNA sequencing data from the SRA database, the gut microbiota composition of CCA patients was compared with that of healthy controls. Mendelian randomization (MR) analysis was employed to assess the genetic association between specific microbiota and CCA risk. Relevant metabolites and genes were obtained from the gutMGene and GeneCards databases for metabolic and functional enrichment analyses. GEO single-cell transcriptomic data (GSE213452) was integrated to characterize the cellular composition of the tumor microenvironment, with a particular focus on T cell subsets and their functional states. The expression differences of key candidate genes were validated using the TCGA-CHOL dataset. Results: Compared with the healthy control group, the gut microbiota composition of CCA patients was significantly altered, characterized by an abnormal enrichment of the phylum Proteobacteria (LDA > 4). MR analysis further demonstrated that genetic predispositions to both the order Enterobacterales and the family Enterobacteriaceae were positively associated with the risk of CCA. Metabolic pathway enrichment analysis suggested that microbiota-related metabolites were mainly involved in pathways such as purine metabolism and glycolysis/gluconeogenesis; functional enrichment analysis showed that related genes were significantly enriched in inflammatory-immune pathways including NOD-like receptor, IL-17, Toll-like receptor, and NF-κB signaling pathways. Single-cell transcriptomic analysis revealed a significantly increased proportion of cancer cells (P < 0.05) and an elevated T cell proportion (from 20.7% to 39.2%) in CCA tissues. Pseudotime analysis indicated that MKI67+ T cells were at a late differentiation stage and exhibited high proliferative features. Differential genes of these T cells intersected with microbiota-related genes, among which SERPINA1 and IFNG showed significant changes in expression in the tumor immune microenvironment (P < 0.001) and may play a core regulatory role. Validation using the TCGA-CHOL dataset revealed that SERPINA1 was significantly downregulated in CCA tumor tissues (P < 0.001), whereas IFNG showed no significant difference between tumor and normal tissues (P > 0.05). Conclusion: Gut microbiota dysbiosis, particularly the abnormal proliferation of Enterobacteriaceae, may promote CCA progression via a metabolic-immune regulatory network. Specifically, alterations in T cell function are closely linked to the differentiated expression patterns of the key genes (SERPINA1 and IFNG) within MKI67+ T cells.