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

作　　者：林清标 林小钦 卓海燕 陈琦 史逸娴 陈丽芳 李东良[2] 王成东 LIN Qing-biao LIN Xiao-qin ZHUO Hai-yan CHEN Qi SHI Yi-xian CHEN Li-fang LI Dong-liang WANG Cheng-dong(Department of Hepatopathy, Mengchao Hepatobiliary Hospital of Fujian Medical Uawersity （ Infectious Disease Hospital Affiliated to Fujian Medical University, Fuzhou Infectious Disease Hospital）, Fuzhou, Fujian 350025, Chin)

机构地区：[1]福建医科大学孟超肝胆医院福建医科大学附属传染病医院福州市传染病医院肝病科,福建福州350025 [2]解放军福州总医院肝胆内科,福建福州350025 [3]连江县医院感染科,福建福州350000

出　　处：《中国临床研究》2017年第9期1153-1156,共4页Chinese Journal of Clinical Research

基　　金：福建省自然科学基金(2012J01399);福州市科技计划项目(2016-S-124-6);福建中医药大学校管科研课题临床专项(XB2015070)

摘　　要：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 mecha

关 键 词：竹节香附素A 人肝癌细胞株Hep G2 血管内皮生长因子 核糖核酸 蛋白 

分 类 号：R735.7[医药卫生—肿瘤]