构树叶总黄酮诱导人肝癌HepG-2细胞凋亡机制探讨  

Mechanism of TFBP for inducing apoptosis of human hepatocellular carcinoma cell line HepG-2

作　　者：朱开梅[1] 李美波[1] 许有瑞[1] 刘建楠[1] 顾生玖[1] 

机构地区：[1]桂林医学院药学院,广西桂林541004

出　　处：《中华肿瘤防治杂志》2014年第18期1413-1418,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：广西科技攻关项目(桂科能129825-21);广西教育厅项目(201202ZD065;2013YB154);桂林市科技攻关项目(20130103-8;20130103-9;20130113-1;20120105-5;20120105-16;20120105-8)

摘　　要：目的:研究构树叶总黄酮(total flavonoids of Broussonetia papyrifera,TFBP)诱导肝癌HepG-2细胞凋亡的机制。方法:采用MTT法检测浓度分别为3、6、9和12g/L TFBP作用于HepG-2细胞24、48和72h后对细胞生长的影响;Hoechst33342荧光染色法观察3、6、9和12g/L TFBP作用于HepG-2细胞48h后细胞形态学的变化;免疫细胞化学检测3、6、9和12g/L TFBP作用于HepG-2细胞48h后凋亡相关基因Bcl-2及Bax蛋白表达;比色法检测3、6、9和12g/L TFBP作用于HepG-2细胞48h后Caspase-3活性。结果:随着药物浓度的增大,TFBP抑制HepG-2细胞增殖的作用逐渐增强,呈剂量依赖性,在3、6、9和12g/L的TFBP作用于HepG-2细胞24h后,HepG-2细胞增殖抑制率分别为7.63%、11.86%、19.02%和21.81%,F=5.16,P=0.034;48h后分别为20.20%、29.44%、39.21%和43.96%,F=11.28,P=0.003;72h后分别为27.24%、34.70%、52.46%和64.72%,F=86.78,P<0.001。Hoechst33342荧光染色法可见,随着药物浓度的增大,细胞的生长变慢,胞质变疏松,细胞核皱缩,深染,出现典型的细胞凋亡形态。免疫细胞化学检测可见,经不同浓度TFBP处理HepG-2细胞48h后,HepG-2细胞Bcl-2蛋白表达随着浓度的增加而逐渐变浅,Bax蛋白表达随着浓度的增加而逐渐变深,Bcl和Bax蛋白的阳性表达率与对照组比较差异均有统计学意义,F值分别为6.563和30.883,P值分别为0.012和0.001。比色法检测Caspase-3活性显示,对照组与3、6、9和12g/L的TFBP对HepG-2细胞作用48h后,Caspase-3酶活性分别为(1.38±2.13)、(3.58±2.21)、(4.95±3.61)、(5.25±1.41)和(5.75±2.24)U/mg,随着TFBP浓度的增加,Caspase-3酶活性显著增高,当TFBP浓度为6、9和12g/L时,各实验组与对照组比较差异有统计学意义,F=5.42,P=0.003。结论:TFBP在体外对肝癌HepG-2细胞有明显的增殖抑制和诱导细胞凋亡的作用。其诱导凋亡的机制可能与其下调Bcl-2蛋白和上调Bax蛋白表达以及增强Caspase-3的活性有关。OBJECTIVE： To study the mechanism of antitumor activity and apoptosis induced by total flavonoids of Broussonetia papyrifera （TFBP） in vitro, using human HepG-2 hepatocellular carcinoma cell line. MElliODS： Different concentrations of TFBP（3,6,9 and 12 g/L） were treated with HepG-2 cell for 24,48 and 72 h and the inhibitory rate of cell proliferation was assessed by MTT method. After 48 h treatment in different concentration of TFBP （3,6,9 and 12 g/L）, Hoechst 33342 fluorescence staining was used to investigate the cells morphological changes. The protein expression of Bcl-2 and Bax was analyzed by immunocytochemical method with 3,6,9 and 12 g/L concentrations of TFBP. Colorimetric method was used to measure Caspase-3 activities. RESULTS： TFBP inhibited HepG-2 cellular proliferation in a dose and time dependent manner （P〈0.05）. Reduction range of HepG-2 cells with different concentration of TFBP treatment （3,6,9 and 12 g/L） after 24 h hours were 7. 63%,11. 86%,19. 02% and 21.81%,F=5. 16,P=0. 034;after 48 hours were 20.20%,29.44%,39.21% and 43.96%,F=11.28,P=0. 003;after 72 hours were 27.24%,34.70%, 52.46% and 64.72%, F= 86. 78, P〈0. 001. With the increasing of drug concentration, cytoplasm loose shrinkage and deep stain of nuclei were observed and nuclcoplasm ratio increased and cells presented typical form apoptosis. TFBPdown-regulated the Bcl-2 expression and up-regulated the Bax expression. F were 6. 563 and 30. 883, P were 0. 012 and 0. 001 respectively. The increase of Caspase-3 activity induced by TFBP showed a concentration-efficiency （F= 5.42, P= 0. 003）. CONCLUSIONS：TFBP has apparent inhibition and apoptosis-inducing effect on HepG-2 cells. TFBP may induce apoptosis via down-regulating Bcl-2 expression, up-regulating of Bax expression, and enhancing Caspase-3 activities in HepG-2 cells.

关 键 词：肝肿瘤 构树叶总黄酮 细胞凋亡 BCL-2 BAX CASPASE-3 

分 类 号：R735.7[医药卫生—肿瘤]