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目的 探讨5-氟尿嘧啶(5-FU)联合低强度超声(LIUS)对人肝癌HepG2细胞杀伤效应及其作用机制。方法 利用LIUS(频率1.0 MHz,强度0.5 W/cm2,占空比20%)联合5-FU作用于人肝癌HepG2细胞系，采用多功能酶标仪测定细胞内5-FU药物含量，MTT法检测肿瘤细胞活力，Annexin V/PI染色、流式细胞术检测细胞凋亡率，偏振光荧光法检测细胞膜的流动性，Western blot法与免疫荧光染色检测核苷转运蛋白1(ENT-1)及P-糖蛋白(P-gp)的表达水平，免疫荧光染色检测钙网蛋白(CRT)和细胞高迁移率族蛋白B1(HMGB1)的表达。结果 LIUS能够显著增强HepG2细胞内5-FU的积累。与无处理组或单独应用5-FU相比，LIUS联合5-FU可显著降低HepG2细胞的活性，增强细胞早期及总细胞凋亡率；显著增加了HepG2细胞膜的流动性；显著上调介导药物转运进入肿瘤细胞的ENT-1的表达并抑制参与药物外排蛋白P-gp的表达；免疫原性细胞死亡(ICD)标志物CRT外翻，HMGB1从细胞核释放到胞质和胞外。结论 LIUS促进5-FU在肝癌HepG2细胞中的药物累积；5-FU联合LIUS通过正向调控药物细胞内转运蛋白ENT-1和负向调控耐药蛋白P-gp的表达，明显增强5-FU对肝癌细胞HepG2的杀伤效应，并诱导细胞免疫原性死亡。
Abstract：Objective To investigate the cytotoxic effects and underlying mechanisms of 5-fluorouracil(5-FU) combined with low-intensity ultrasound(LIUS) in human hepatocellular carcinoma HepG2 cells. Methods HepG2 cells were treated with LIUS(1.0 MHz, 0.5 W/cm2, duty cycle 20%) in combination with 5-FU. Intracellular 5-FU accumulation was quantified using a fluorescence microplate reader. Cell viability was assessed using an MTT assay, and apoptosis was measured by Annexin V/PI staining followed by flow cytometry. Membrane fluidity was assessed by fluorescence polarization. The expression levels of equilibrative nucleoside transporter 1(ENT-1) and P-glycoprotein(P-gp) were detected by Western blot and immunofluorescence. Immunofluorescence staining was further used to evaluate the expression and translocation of immunogenic cell death(ICD) markers, calreticulin(CRT), and high mobility group protein B1(HMGB1). Results LIUS significantly enhanced the intracellular accumulation of 5-FU in HepG2 cells. Compared with untreated cells or those treated with 5-FU alone, the combination of LIUS and 5-FU significantly reduced cell viability and increased early and total apoptosis. Membrane fluidity was significantly increased in the combination group. Additionally, the expression of ENT-1, which mediated drug influx, was significantly upregulated, while the expression of the drug efflux protein P-gp was markedly downregulated.CRT translocated to the cell surface, and HMGB1 was released from the nucleus to the cytoplasm and extracellular space, indicating the induction of ICD. Conclusion LIUS enhances the intracellular accumulation of 5-FU in HepG2 cells. The combination of 5-FU and LIUS exerts potent cytotoxic effects by upregulating ENT-1 and downregulating P-gp, and induces immunogenic cell death in hepatocellular carcinoma cells.
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基本信息:
DOI：10.13423/j.cnki.cjcmi.010088
中图分类号:R735.7
引用信息:
[1]鲁毅,张玮函,杨馥瑜,等.低强度超声联合5-氟尿嘧啶对人肝癌HepG2细胞的抑制作用及机制[J].细胞与分子免疫学杂志,2026,42(04):299-306.DOI:10.13423/j.cnki.cjcmi.010088.
基金信息:
国家自然科学基金资助项目(81703040); 装备综合科学研究项目(KJ2023C001-D029)
2025-12-01
2025-12-01
2025-12-01