竹节香附素A对人肝癌HepG_2细胞VEGF基因表达的影响

Effect of Raddeanin A on the expression of vascular endothelial growth factor gene in human hepatocellular carcinoma HepG_2 cells

目的观察竹节香附素A对人肝癌细胞株HepG_2细胞血管内皮生长因子(VEGF)表达的影响。方法体外培养HepG_2细胞,将不同浓度的竹节香附素A与HepG_2细胞共培养,采用酶联免疫吸附(ELISA)方法检测不同浓度竹节香附素A处理HepG_2细胞24 h后细胞培养上清液中VEGF蛋白含量的变化;半定量逆转录-聚合酶链反应(RT-PCR)法和蛋白免疫印迹(Western blot)法分别检测竹节香附素A对HepG_2细胞VEGF mRNA和蛋白相对表达水平(阳性条带灰度值/β-actin带灰度值)的影响。结果 (1)不同浓度(5、10、20μg/ml)竹节香附素A处理HepG_2细胞24 h后,细胞培养上清液中VEGF水平呈剂量依赖性降低(P=0.000)。(2)竹节香附素A对HepG_2细胞VEGF mRNA表达的影响:(1)不同浓度竹节香附素A(0、5、10、20μg/ml)处理HepG_2细胞24 h后,VEGF mRNA的相对表达量呈剂量依赖性减少(0.96±0.07、0.75±0.09、0.55±0.10、0.32±0.11,P=0.000)。(2)10μg/ml竹节香附素A分别处理HepG_2细胞0、6、12、24 h后,VEGF mRNA的相对表达水平呈时间依赖性下降(0.77±0.05、0.53±0.09、0.32±0.12、0.15±0.04,P=0.000)。(3)竹节香附素A抑制HepG_2细胞VEGF蛋白的表达:(1)不同浓度竹节香附素A(0、5、10、20μg/ml)处理HepG_2细胞24 h后,VEGF蛋白相对表达水平呈剂量依赖性下降(2.08±0.28、1.43±0.22、0.72±0.15、0.17±0.10,P=0.000)。(2)10μg/ml竹节香附素A分别处理HepG_2细胞0、6、12、24 h后VEGF蛋白相对表达水平呈时间依赖性减少(2.47±0.05、1.36±0.02、0.30±0.02、0.07±0.02,P=0.000)。结论竹节香附素A能够下调HepG_2细胞VEGF表达,这可能是其抑制肝癌细胞生长增殖的重要机制之一。

Objective To observe the effect of Raddeanin A on the expression of vascular endothelial growth factor （VEGF） gene in human hepatocellular carcinoma HepG2 cells. Methods HepG2 cells were cultured with different concentrations of Raddeanin A in vitro. Enzyme-linked immuno sorbent assay （ ELISA ） was used to detect VEGF level in cell culture supernatant after HepG2 cells treated with different concentration of Raddeanin A for 24 h. Reverse transeription-polymerase chain reaction（RT-PCR） and protein immunoblot （western blot） were used to detect the relative expression level of messenger ribonucleic acid （mRNA） and protein （ the ratio of gray scale value of the positive band to the β-actin） of VEGF in HepG2 cells respectively. Results （ 1 ） After HepG2 cells were treated by different concentrations （5,10 and 20μg/ml） of Raddeanin A, the level of VEGF protein in cell culture supernatant decreased in a dose dependent manner（P = 0. 000）. （2） The relative expression level of VEGF mRNA decreased in a dose dependent manner after HepG2 cells treated with 0,5,10 and 20μg/ml Raddcanin A for 24 h （0.96±0.07 vs 0. 75±0. 09 vs 0. 55±0. 10 vs 0.32±0.11, P = 0. 000）. The relative expression level of VEGF mRNA decreased in a time dependent manner after HepG2 cells treated with 10 μg/ml Raddeanin A for 0,6,12 and 24 h （0.77 ±0.05 vs 0. 53 ±0.09 vs 0. 32±0.12 vs 0. 15±0.04 ,P = 0. 000）. （3） The relative expression level of VEGF protein decreased in a dose dependent manner after HepG2 cells treated with 0,5,10 and 20μg/ml Raddeanin A for 24 h （2.08 ±0.28 vs 1.43 ±0.22 vs 0. 72 ±0.15 vs 0. 17 ± 0.10,P = 0.000）. The relative expression level of VEGF protein decreased in a time dependent manner after HepG2 cells treated with 10 μg/ml Raddeanin A for 0,6,12 and 24 h （2.47 ±0.05 vs 1.36 ±0.02 vs 0. 30 ±0.02 vs 0. 07 ± 0. 02,P=0. 000）. Conclusion Raddeanin A could down-regulate the expression of VEGF gene in HepG2 cells, which may be one of the important mechanisms to inhibit the growth and proliferation of liver cancer cells.