[摘 要] 目的：探讨蟾毒灵(BU)对肿瘤相关巨噬细胞(TAM)介导的结直肠癌(CRC)细胞程序性死亡蛋白-配体1(PD-L1)表达的调控作用及其分子机制。方法：采用HCT116细胞与THP-1来源巨噬细胞构建体外共培养体系。佛波酯(PMA)诱导THP-1细胞分化为M0型巨噬细胞，进一步以HCT116细胞条件培养基(CM)刺激形成TAM样细胞。流式细胞术检测CD11b、CD206表达，以评价巨噬细胞极化水平；RT-qPCR及WB法检测TGF-β、IL-10、STAT3/p-STAT3及PD-L1表达变化；慢病毒转染构建STAT3敲低细胞系HCT116-shSTAT3，结合BU干预验证STAT3/PD-L1信号通路作用。结果：HCT116细胞CM可诱导巨噬细胞向M2表型极化，表现为CD11b+CD206+细胞比例升高（P < 0.01）及TGF-β、IL-10表达增加（均P < 0.001）。TAM条件培养基可显著促进HCT116细胞STAT3磷酸化（P < 0.001）并上调PD-L1 mRNA及蛋白表达水平（P < 0.001或P < 0.01）。BU干预后，TAM介导的STAT3磷酸化水平明显下降，PD-L1表达同步下调（均P < 0.01）。STAT3敲低可降低PD-L1表达，其作用趋势与BU一致。结论：BU可通过抑制TAM介导的STAT3信号激活，下调CRC细胞PD-L1表达，提示其在肿瘤免疫微环境中具有潜在的应用价值。

[Abstract] Objective: To investigate the regulatory effect of bufalin (BU) on tumor-associated macrophage (TAM)-mediated programmed death-ligand 1 (PD-L1) expression in colorectal cancer (CRC) cells and to elucidate the underlying molecular mechanism. Methods: An in vitro co-culture system was established using HCT116 cells and THP-1-derived macrophages. THP-1 cells were differentiated into M0 macrophages by PMA treatment and further stimulated with HCT116-conditioned medium (CM) to generate TAM-like cells. Flow cytometry was used to detect CD11b and CD206 expressions and assess macrophage polarization level. The changes in the expression levels of TGF-β, IL-10, STAT3/p-STAT3, and PD-L1 were detected by RT-qPCR and WB assay. A STAT3 knockdown cell line (HCT116-shSTAT3), constructed by lentiviral transduction, was combined with BU treatment to verify the role of the STAT3/PD-L1 signaling pathway. Results: HCT116-derived CM induced macrophage polarization toward the M2 phenotype, as evidenced by an increased proportion of CD11b+CD206+ cells (P < 0.01) and elevated expression of TGF-β and IL-10 (both P < 0.001). TAM-conditioned medium significantly promoted STAT3 phosphorylation (P < 0.001) and upregulated PD-L1 expression at both mRNA and protein levels in HCT116 cells (P < 0.001 or P < 0.01). BU treatment markedly suppressed TAM-mediated STAT3 phosphorylation and concomitantly reduced PD-L1 expression (both P < 0.01). STAT3 knockdown decreased PD-L1 expression, with a trend consistent with BU treatment. Conclusion: BU can downregulate PD-L1 expression in CRC cells by inhibiting TAM-mediated STAT3 signaling activation, suggesting its potential application value in the tumor immune microenvironment.

上海市卫生健康系统重点学科（2024ZDXK0044，2024ZDXK0046）；上海市普陀区卫生健康系统科技创新项目