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目的:研究甘草查尔酮B(licochalcone B)诱导小鼠黑色素瘤细胞(B16F0)凋亡作用并初步探讨其机制。方法:取对数生长期B16F0细胞按5 000个/孔接种于96孔板中,MTT染色法检测甘草查尔酮B(5,7.5,10,12.5,15 mg.L-1)作用24 h后,对B16F0细胞的增殖抑制作用;取对数生长期B16F0细胞按1×105个/孔接种于6孔板中,甘草查尔酮B(7.5,15mg.L-1)作用24 h后,Hoechst 33258染色法观察细胞凋亡形态;取对数生长期B16F0细胞按5×105个/瓶接种于细胞培养瓶中,甘草查尔酮B(5,7.5,10,12.5 mg.L-1)作用24 h后,流式细胞仪检测细胞凋亡率;RT-PCR法检测与凋亡相关基因Bcl-2相关X蛋白(Bax),B淋巴细胞瘤-2(Bcl-2)mRNA表达;荧光法检测半胱氨酸蛋白酶蛋白9(Caspase-9)和半胱胺酸蛋白酶蛋白3(Caspase-3)相对活性。结果:甘草查尔酮B能有效抑制B16F0细胞增殖,作用24 h后对细胞的生长有明显的抑制作用,IC5011.3 mg.L-1,在5～15 mg.L-1表现出明显的剂量依赖,细胞出现明显凋亡特征,随甘草查尔酮B浓度的增加凋亡率逐渐升高;甘草查尔酮B(10,12.5 mg.L-1)能明显下调Bcl-2/Bax,上调Caspase-3,Caspase-9的表达,其中7.5 mg.L-1甘草查尔酮B引起Caspase-9,Caspase-3的活化程度较12.5 mg.L-1高。结论:甘草查尔酮B可能通过激活线粒体调控的凋亡通路诱导B16F0细胞凋亡。
Objective: To study the apoptosis of melanoma cells (B16F0) induced by licochalcone B and its mechanism. Methods: B16F0 cells in logarithmic growth phase were inoculated into 96-well plates at 5 000 cells / well, and the effects of licochalcone B (5, 7.5, 10, 12.5, 15 mg.L-1) (B16F0 cells in logarithmic growth phase) were seeded in 6-well plates at a density of 1 × 105 cells / well and treated with licochalcone B (7.5, 15 mg.L-1) for 24 h. Hoechst 33258 staining was used to observe the morphology of apoptotic cells. B16F0 cells in logarithmic growth phase were inoculated in 5 × 105 cells / flask in a cell culture flask. Licochalcone B (5, 7.5, 10, 12.5 mg.L-1) The apoptosis rate was detected by flow cytometry after 24 h of treatment. The expression of Bcl-2 and Bcl-2 mRNA was detected by RT-PCR. The fluorescence Method to detect the relative activity of Caspase-9 and Caspase-3. Results: Licochalcone B could effectively inhibit the proliferation of B16F0 cells, and had a significant inhibitory effect on the growth of B16F0 cells after 24 h. IC5011.3 mg.L-1 at 5-15 mg.L-1 showed obvious The apoptotic rate increased with the increase of concentration of licochalcone B. The apoptosis rate of licorice chalcone B (10, 12.5 mg.L-1) could significantly down-regulate Bcl-2 / Bax, Upregulation of Caspase-3, Caspase-9 expression, of which 7.5 mg.L-1 licochalcone B caused Caspase-9, Caspase-3 activation than 12.5 mg.L-1 high. Conclusion: Licochalcone B may induce apoptosis of B16F0 cells by activating the mitochondria-regulated apoptotic pathway.