目的·明确CCCTC结合因子（CCCTC-binding factor，CTCF）对肝细胞脂质代谢的调控作用，并探究CTCF调控肝细胞基因表达的作用机制。方法·通过慢病毒将稳定表达Ctcf shRNA的DNA序列整合到小鼠永生化的AML12肝细胞系中，实现对Ctcf的稳定敲低。通过反转录实时荧光定量聚合酶链反应（RT-qPCR）及蛋白质印迹法（Western blotting）验证Ctcf的敲低效率。碘化丙啶（propidium iodide，PI）染色测定细胞周期，使用CCK-8法绘制细胞生长曲线，检测Ctcf敲低对细胞生长的影响。油红O染色标记细胞内脂质，检测CTCF对AML12细胞脂质代谢和脂滴累积的影响。使用CUT & Tag测序技术分析Ctcf敲低后CTCF在全基因组的结合变化。结合RNA测序（RNA-seq）的转录组数据分析CTCF结合变化后的转录组变化，使用基因本体论（GO）、京都基因和基因组数据库（KEGG）富集分析及基因集富集分析（GSEA）揭示Ctcf敲低对AML12肝细胞基因的表达影响，并探究差异基因与CTCF结合变化间的关联。结果·RT-qPCR结果表明Ctcf在 mRNA水平敲低了63.4%，Western blotting验证了CTCF在蛋白表达层面降低了57.7%（均P<0.05）。生长曲线及周期实验确定了Ctcf敲低后细胞增殖阻滞于G₁/G₀期。并且AML12细胞在Ctcf敲低后自发出现细胞内脂质蓄积（P<0.05）。CTCF在全基因组的结合呈现出显著变化，大多数CTCF结合差异区域表现出CTCF结合减少，但仍有部分区域CTCF结合增加。转录组数据显示Ctcf敲低导致1 344个基因出现显著的表达变化，在上调基因中富集出与脂滴堆积相关的脂质代谢通路。CTCF结合上调峰关联的差异基因富集在脂质转运与脂质定位相关通路，而CTCF结合下调峰关联的差异基因主要富集在DNA损伤修复、细胞凋亡、细胞周期等生物学过程，但CTCF在基因组的结合变化并不足以导致邻近基因的表达上调或下调。结论·CTCF通过调控脂质代谢相关基因的表达影响肝细胞的代谢功能，然而CTCF在基因组上的结合变化与邻近基因的表达缺乏显著的相关性，可能主要通过远端调控的方式对基因表达产生影响。

Objective ·To clarify the regulatory role of CCCTC-binding factor (CTCF) in lipid metabolism in liver cells, and explore the mechanisms by which CTCF regulates liver cell gene expression. Methods ·Immortalized AML12 liver cell line was used as a model to investigate the functions of CTCF in liver cells. To stably knock down Ctcf, DNA sequences stably expressing Ctcf shRNA were integrated into AML12 cells through lentivirus. The knockdown efficiency of Ctcf was verified by RT-qPCR and Western blotting. The effects of Ctcf knockdown on cell growth and cell cycle were assessed by performing CCK-8 assay and propidium iodide (PI) staining. Intracellular lipids, labeled with Oil Red O staining, were analyzed and quantified to detect the effect of CTCF on lipid metabolism and lipid droplet accumulation in AML12 cells. Changes in CTCF genome distribution after Ctcf knockdown were analyzed using the Cleavage Under Targets and Tagmentation (CUT&Tag) method. Transcriptome changes in AML12 cells after Ctcf knockdown were quantified by RNA sequencing (RNA-seq). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and gene set enrichment analysis (GSEA) were employed to evaluate the functions of differentially expressed genes. The correlation between gene expression changes and CTCF binding changes was further assessed by performing statistical analyses. Results ·The result of RT-qPCR showed that Ctcf is downregulated 63.4% in mRNA level and 57.7% in protein level (both P<0.05). Assay of the growth curve and cycle phase confirmed that cell proliferation was inhibited in the G₁/G₀ phase after Ctcf knockdown. After Ctcf knockdown, AML12 cells exhibited spontaneous accumulation of intracellular lipids, indicating dysregulation of lipid metabolism (P<0.05). Genome-wide CTCF binding analysis revealed significant changes, with most differential CTCF peaks showing decreased binding, although a subset of regions exhibited increased CTCF binding. Transcriptome analyses revealed that knocking down Ctcf resulted in significant expression changes in 1 344 genes. These differentially expressed genes were enriched in lipid metabolism pathways. Further analysis showed that genes associated with regions of increased CTCF binding were enriched in pathways related to lipid transport and localization, whereas genes associated with regions of decreased CTCF binding were mainly enriched in processes such as DNA damage repair, apoptosis, and cell cycle regulation. However, the binding changes of CTCF in the genome were not sufficient to lead to the expression changes of their neighboring genes. Conclusion ·CTCF affects the metabolic function of liver cells by regulating the expression of lipid metabolism-related genes. However, the binding changes of CTCF in the genome lack significant correlation with the expression of their neighboring genes, suggesting that CTCF mainly influences liver gene expression through long-distance regulation, possibly by modulating higher-order chromatin structure and enhancer-promoter interactions.