【摘要】 目的 通过16S rRNA高通量测序技术和类器官模型，探讨肝棘球蚴抗原处理对肝外胆管癌(CCA)类器官生态微环境的影响，揭示肝棘球蚴在CCA发病机制中的潜在作用。方法 本研究为前瞻性对照研究，纳入60例肝外胆管癌(CCA)的患者，分为实验组(暴露于肝棘球蚴抗原)和对照组(暴露于PBS),每组30例样本。通过类器官技术培养肿瘤组织样本，并使用16S rRNA高通量测序技术分析2组样本的微生物群落组成和功能基因丰度。使用线性判别分析效应量(LEfSe)方法和PICRUSt工具进行微生物群落差异分析和功能预测分析，进一步通过qPCR和细胞功能实验验证关键微生物类群和信号通路的变化。结果 实验组和对照组的主要微生物门包括厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和耐热菌门(Thermotogae)。实验组中厚壁菌门和放线菌门的丰度显著高于对照组，而拟杆菌门的丰度显著降低(P<0.05)。α多样性分析显示，实验组的Chao1指数(150.25±15.75)和Shannon指数(3.50±0.45)均显著高于对照组(Chao1指数：140.80±14.95,Shannon指数：3.31±0.40,P<0.05)。β多样性分析基于Bray-Curtis距离的主坐标分析(PCoA)和非度量多维尺度分析(NMDS)结果显示，实验组和对照组在微生物群落组成上存在显著差异(PERMANOVA:F=2.423,P<0.001)。PICRUSt分析结果显示，实验组在碳水化合物代谢(25.30%±2.50%vs 23.10%±2.40%)、能量代谢(18.75%±1.95%vs 17.50%±2.10%)、脂类代谢(15.20%±1.80%vs 14.00%±1.75%)、氨基酸代谢(12.50%±1.60%vs 11.20%±1.55%)、核苷酸代谢(10.00%±1.25%vs 9.00%±1.20%)和信号转导(8.75%±1.10%vs 7.50%±1.05%)等功能路径的基因丰度上显著高于对照组(P<0.05)。qPCR结果显示，实验组中厚壁菌门(t=2.191,P=0.033)、变形菌门(t=2.253,P=0.029)和放线菌门(t=2.311,P=0.026)的相对丰度显著增加，而拟杆菌门(t=2.432,P=0.021)和耐热菌门(t=2.732,P=0.009)的相对丰度显著降低。细胞功能实验显示，实验组在细胞增殖、迁移和侵袭能力上显著增强(P<0.05)。Spearman相关性分析表明厚壁菌门的丰度与患者年龄(r=0.452,P=0.001)和病史(r=0.322,P=0.013)呈显著正相关，与肿瘤分级呈显著负相关(r=-0.455,P=0.001),而与肿瘤分期呈显著正相关(r=0.311,P=0.024)。结论 本研究通过高通量测序和功能预测分析，揭示了肝棘球蚴抗原处理对肝外胆管癌类器官微生态环境的显著影响，尤其体现在微生物群落组成和功能基因丰度的改变。更多还原

【Abstract】 Objective To investigate the effect of hydatid antigen treatment on the ecological microenvironment of extrahepatic cholangiocarcinoma(CCA) organoids by using 16S rRNA high-throughput sequencing technology and organoid models, and to reveal the potential role of hydatid cysts in the pathogenesis of CCA. Methods This study was a prospective controlled study, in which 60 patients diagnosed with extrahepatic cholangiocarcinoma(CCA) were enrolled and divided into an experimental group(exposed to hydatid antigen) and a control group(exposed to PBS),with 30 samples in each group. Tumor tissue samples were cultured by organoid technology, and 16S rRNA high-throughput sequencing technology was used to analyze the microbial community composition and functional gene abundance of the two groups of samples. The linear discriminant analysis effect size(LEfSe) method and PICRUSt tool were used for microbial community difference analysis and functional prediction analysis, and the changes of key microbial groups and signaling pathways were further verified by qPCR and cell function experiments. Results The main microbial phyla in the experimental and control groups included Firmicutes,Bacteroidetes,Proteobacteria,Actinobacteria,and Thermotogae. The abundance of Firmicutes and Actinobacteria in the experimental group was significantly higher than that in the control group, while the abundance of Bacteroidetes was significantly decreased(P<0.05). α-diversity analysis showed that the Chao1 index(150.25±15.75) and Shannon index(3.50±0.45) in the experimental group were significantly higher than those in the control group(Chao1 index: 140.80±14.95,Shannon index: 3.31±0.40,P<0.05). The results of principal coordinates analysis(PCoA) and nonmetric multidimensional scaling(NMDS) based on Bray-Curtis distance for β-diversity analysis showed that there were significant differences in the composition of the microbial communities between the experimental and control groups(PERMANOVA:F=2.423,P<0.001). PICRUSt analysis results showed that the gene abundance of the experimental group in functional pathways such as carbohydrate metabolism(25.30%±2.50% vs 23.10%±2.40%),energy metabolism(18.75%±1.95% vs 17.50%±2.10%),lipid metabolism(15.20%±1.80% vs 14.00%±1.75%),amino acid metabolism(12.50%±1.60% vs 11.20%±1.55%),nucleotide metabolism(10.00%±1.25% vs 9.00%±1.20%) and signal transduction(8.75%±1.10% vs 7.50%±1.05%) was significantly higher than that in the control group(P<0.05). qPCR results showed that the relative abundance of Firmicutes(t=2.191,P=0.033),Proteobacteria(t=2.253,P=0.029) and Actinobacteria(t=2.311,P=0.026) in the experimental group increased significantly, while the relative abundance of Bacteroidetes(t=2.432,P=0.021) and Thermotolerant bacteria(t=2.732,P=0.009) decreased significantly. Cell function experiments showed that the experimental group had significantly enhanced cell proliferation, migration and invasion abilities(P<0.05). Spearman correlation analysis showed that the abundance of Firmicutes was significantly positively correlated with patient age(r=0.452,P=0.001) and medical history(r=0.322,P=0.013),and was significantly negatively correlated with tumor grade(r=-0.455,P=0.001),and was significantly positively correlated with tumor stage(r=0.311,P=0.024). Conclusion This study revealed the significant effect of hydatid antigen treatment on the microecological environment of extrahepatic cholangiocarcinoma organoids through high-throughput sequencing and functional prediction analysis, especially in the changes of microbial community composition and functional gene abundance.更多还原