目的 探討橙皮苷通過(guò)靶向β-連環(huán)蛋白（β-catenin）對(duì)肝癌細(xì)胞干性的抑制作用，評(píng)估其對(duì)索拉非尼敏感性的增強(qiáng)效果。方法 取對(duì)數(shù)生長(zhǎng)期的HepG2細(xì)胞，設(shè)置對(duì)照組和橙皮苷低、高劑量（0.5、1.5 mmol/L）組，進(jìn)行腫瘤成球?qū)嶒?yàn)，采用Western blotting和免疫熒光技術(shù)檢測(cè)干性相關(guān)蛋白表達(dá)，采用qRT-PCR檢測(cè)干性相關(guān)基因表達(dá)。HepG2細(xì)胞培養(yǎng)1周后，測(cè)定細(xì)胞對(duì)索拉非尼的半數(shù)抑制濃度（half inhibitory concentration，IC₅₀）曲線，觀察細(xì)胞增殖與凋亡情況。構(gòu)建穩(wěn)定過(guò)表達(dá)β-catenin的HepG2細(xì)胞系，設(shè)置過(guò)表達(dá)β-catenin組和過(guò)表達(dá)β-catenin＋橙皮苷（1.5 mmol/L）組，進(jìn)行腫瘤成球?qū)嶒?yàn)，并檢測(cè)干性相關(guān)蛋白表達(dá)；測(cè)定細(xì)胞對(duì)索拉非尼的IC₅₀曲線，觀察細(xì)胞增殖與凋亡情況。裸鼠注射HepG2細(xì)胞后，給予橙皮苷或索拉非尼治療，定期觀察并記錄瘤體大小，采用免疫組化與Western blotting檢測(cè)腫瘤組織Ki67、β-catenin、半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）、CD44、CD133蛋白表達(dá)。結(jié)果 橙皮苷能有效抑制腫瘤細(xì)胞成球能力（P＜0.001），減少細(xì)胞球體的體積和數(shù)量（P＜0.001），并顯著降低干性相關(guān)蛋白表達(dá)（P＜0.001）。此外，橙皮苷可增強(qiáng)腫瘤細(xì)胞對(duì)索拉非尼的敏感性，影響β-catenin的定位。體內(nèi)實(shí)驗(yàn)結(jié)果顯示，橙皮苷具有抑瘤作用，與索拉非尼聯(lián)用時(shí)效果更佳。結(jié)論 橙皮苷通過(guò)靶向β-catenin，抑制肝癌細(xì)胞干性，并顯著增強(qiáng)了索拉非尼對(duì)肝癌細(xì)胞的敏感性。

Objective To explore the inhibitory effect of hesperidin on stemness of hepatoma cells by targeting β-catenin, and evaluate its enhancing effect on sorafenib sensitivity. Methods HepG2 cells in logarithmic growth phase were selected, and control group, hesperidin low-, high-dose (0.5, 1.5 mmol/L) groups were set up for tumor spheroidization experiments, Western blotting and immunofluorescence techniques were used to detect the expressions of stemness related proteins, and qRT PCR was used to detect the expressions of stemness related genes. After one week of HepG2 cells culture, the half inhibitory concentration (IC₅₀) curve of cells against sorafenib was measured to observe cell proliferation and apoptosis. A stable HepG2 cell line overexpressing β-catenin was constructed, overexpressing β-catenin group and overexpressing β-catenin + hesperidin (1.5 mmol/L) group were set up for tumor spheroidization experiments, and the expressions of stemness related proteins were detected; The IC₅₀ curve of cells against sorafenib was measured to observe cell proliferation and apoptosis. After injecting HepG2 cells into nude mice, hesperidin or sorafenib was administered, tumor size was regularly observed and recorded. Immunohistochemistry and Western blotting were used to detect the expressions of Ki67, β-catenin, cysteine aspartate protease-3 (Caspase-3), CD44 and CD133 proteins in tumor tissue. Results Hesperidin could effectively inhibit the spheroidization ability of tumor cells (P < 0.001), reduce the volume and quantity of cell spheroids (P < 0.001), and significantly decrease the expressions of stemness related proteins (P < 0.001). In addition, hesperidin could enhance the sensitivity of tumor cells to sorafenib and affect the localization of β-catenin. The in vivo experimental results showed that hesperidin had anti-tumor effects, and the combination with sorafenib had a better effect. Conclusion Hesperidin inhibits the stemness of hepatoma cells by targeting β-catenin, and significantly enhances the sensitivity of sorafenib to hepatoma cells.

R285.5

山東省中醫(yī)藥科技面上項(xiàng)目（M-2023215）