目的 探討橙皮苷通過靶向β-連環(huán)蛋白（β-catenin）對肝癌細胞干性的抑制作用，評估其對索拉非尼敏感性的增強效果。方法 取對數(shù)生長期的HepG2細胞，設置對照組和橙皮苷低、高劑量（0.5、1.5 mmol/L）組，進行腫瘤成球?qū)嶒?，采用Western blotting和免疫熒光技術檢測干性相關蛋白表達，采用qRT-PCR檢測干性相關基因表達。HepG2細胞培養(yǎng)1周后，測定細胞對索拉非尼的半數(shù)抑制濃度（half inhibitory concentration，IC₅₀）曲線，觀察細胞增殖與凋亡情況。構建穩(wěn)定過表達β-catenin的HepG2細胞系，設置過表達β-catenin組和過表達β-catenin＋橙皮苷（1.5 mmol/L）組，進行腫瘤成球?qū)嶒?，并檢測干性相關蛋白表達；測定細胞對索拉非尼的IC₅₀曲線，觀察細胞增殖與凋亡情況。裸鼠注射HepG2細胞后，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。結果 橙皮苷能有效抑制腫瘤細胞成球能力（P＜0.001），減少細胞球體的體積和數(shù)量（P＜0.001），并顯著降低干性相關蛋白表達（P＜0.001）。此外，橙皮苷可增強腫瘤細胞對索拉非尼的敏感性，影響β-catenin的定位。體內(nèi)實驗結果顯示，橙皮苷具有抑瘤作用，與索拉非尼聯(lián)用時效果更佳。結論 橙皮苷通過靶向β-catenin，抑制肝癌細胞干性，并顯著增強了索拉非尼對肝癌細胞的敏感性。

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)藥科技面上項目（M-2023215）