构树叶总黄酮调控Bcl-2与Bax蛋白表达及caspase-3活性诱导HepG-2细胞凋亡的研究

Effect of Total Flavonoids of Broussonetia Papyrifera on Apoptosis of Human Hepatocellular Carcinoma Cell Line HepG-2

目的:研究构树叶总黄酮(total flavonoids of Broussonetia papyrifera,TFBP)诱导HepG-2细胞凋亡的机制。方法:采用体外细胞培养方法,取对数生长期人肝癌HepG-2细胞,随机分为药物组和对照组,药物组用3,6,9,12 g·L-1不同质量浓度TFBP作用人肝癌HepG-2细胞24,48,72 h后,应用MTT法检测TFBP对人肝癌细胞HepG-2生长抑制作用;Hoechst 33342荧光染色法荧光显微镜观察各浓度TFBP作用细胞48 h后的细胞的形态变化;通过免疫细胞化学SP法检测凋亡相关基因B细胞淋巴瘤/白血病2(Bcl-2),Bcl-2相关X蛋白(Bax)蛋白表达;比色法检测半胱氨酸蛋白酶-3(caspase-3)活性。结果:TFBP在体外对人肝癌细胞HepG-2有较强的浓度依赖性和时间依赖性抑制作用,9,12 g·L-1的TFBP作用HepG-2细胞72 h后,细胞增殖抑制率可分别达52.46%,64.72%,与对照组具有显著性差异(P<0.01),可诱导细胞凋亡,质量浓度9,12 g·L-1的TFBP作用HepG-2细胞48 h后就可出现典型的凋亡小体,TFBP可升高caspase-3活性,并具有浓度效应,同时还可下调Bcl-2蛋白表达,上调Bax蛋白表达,下调Bcl-2/Bax。结论:TFBP在体外对肝癌细胞HepG-2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蛋白的表达,下调Bcl-2/Bax以及增强caspase-3的活性有关。

Objective： The total flavonoids of Broussonetia papyrifera （TFBP） was investigated for its antitumor activity and induction of apoptosis in vitro, using human HepG-2 hepatocellular carcinoma cell line. Method： The hepatoeellular carcinoma HepG-2 cells in logarithmic phase of growth were randomly divided into drug group and control group, with the concentration of 3, 6, 9, 12 g ·L^-1 TFBP in hepatocellular carcinoma HepG-2 cells intervention durations were 24, 48, 72 h accordingly. The viability of HepG-2 cells was measured by MTT. Morphology of cell apoptosis was observed by Hoechst 33342 fluorescence staining. The protein expression of B cell lymphoma/leukemia-2 （Bcl-2） and Bcl-2 associated X protein （Bax） was analyzed by immunocytochemical method （SP）. Result： TFBP inhibited the growth of cells and caused apoptosis significantly. The suppression was both in a time-and dose-dependent manner. When HepG-2 cells were treated with the concentration of 9, 12 g ·L^-1 TFBP in 72 h, the inhibition rate can be achieved 52.46% , 64.72% respectively. Compared with control group, there has significant difference （P 〈 0.01 ）. When HepG-2 cells. were treated with the concentration of 9, 12 g ·L^-1 TFBP in 48 h, the typical apoptotic body can be observed under the fluorescence microscope. The increase of caspase-3 activity induced by TFBP showed a concentrationefficiency. TFBP down-regulated the Bcl-2 expression and up-regulated the Bax expression. The ratio of Bcl-2/Bax was evident decrease. Conclusion： 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, decreasing the ratio of Bcl-2/Bax and enhancing caspase-3 activities in HepG-2 cells.