目的 研究华蟾毒配基联合索拉非尼对肝癌Huh7细胞增殖与凋亡的影响及作用机制。方法 MTT法检测Huh7细胞增殖；Hoechst33342/PI荧光双染法检测Huh7细胞凋亡形态变化；流式细胞仪检测细胞周期；免疫细胞化学法检测Ki67蛋白表达；Western blotting法检测Bax、Bcl-2、Caspase-8、极光激酶A（AURKA）、Ras、Raf、细胞外调节蛋白激酶（ERK）和p-ERK蛋白的表达变化。结果 华蟾毒配基、索拉非尼及联合用药对Huh7细胞的增殖均有抑制作用，联合用药组的增殖抑制作用更明显，具有协同效应；荧光染色可见细胞凋亡形态变化；索拉非尼引起Huh7细胞周期S期阻滞，华蟾毒配基和联合用药引起Huh7细胞周期G₂/M期阻滞，联合用药组G₂/M期阻滞较单药组更明显；华蟾毒配基和索拉非尼均能减弱Ki67表达，联合用药组的作用更为显著；华蟾毒配基、索拉非尼及联合用药上调Bax和Caspase-8蛋白表达，下调Bcl-2蛋白表达，上调Bax/Bcl-2比例，对ERK蛋白表达无明显影响，显著下调AURKA、Ras、Raf和p-ERK蛋白表达，联合用药组的作用更为显著（P<0.05）。结论 华蟾毒配基联合索拉非尼通过AURKA/Ras/Raf/ERK信号通路抑制肝癌Huh7细胞增殖，诱导其凋亡。

Objective To investigate the effect and mechanism of cinobufagin combined with Sorafenib on the proliferation and apoptosis of hepatocellular carcinoma Huh7 cells. Methods The proliferation of Huh7 cells was measured using MTT assay; The apoptosis morphological changes of Huh7 cells were detected using Hoechst33342/PI fluorescence staining; The cells cycle was detected by flow cytometry; The expression of Ki67 protein was detected by immunocytochemistry; The expressions of Bax, Bcl-2, Caspase-8, AURKA, Ras, Raf, ERK, and p-ERK proteins were measured using Western blotting. Results Cinobufagin, Sorafenib, and combination therapy inhibited the proliferation of Huh7 cells, and the inhibitory effect of the combination group was more obvious with synergistic effect. Fluorescence staining showed morphological changes of apoptosis. Sorafenib induced the cell cycle S phase arrest, cinobufagin and combination therapy induced the cell cycle G₂/M phase arrest, combination group had more obvious cell cycle arrest in G₂/M phase than single drug groups. Both cinobufagin and Sorafenib attenuated the expression of Ki67, and the effect of combination group was more significant. Cinobufagin, Sorafenib, and combination therapy up-regulated the expression of Bax and Caspase-8 proteins; down-regulated the expression of Bcl-2 protein; up-regulated the ratio of Bax/Bcl-2; had no obvious effect on the expression of ERK protein; significantly down-regulated the expression of AURKA, Ras, Raf, and p-ERK proteins; And the effect of combination group was more significant (P < 0.05). Conclusion Cinobufagin combined with Sorafenib could inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma Huh7 cells through AURKA/Ras/Raf/ERK signaling pathway.

国家自然科学基金资助项目（81673651，81273552，81273745）；天津市自然科学基金重点项目（I8JCZDJC36500）；武警后勤学院博士启动金项目（WHB201501）