构树叶总黄酮诱导人肝癌HepG-2细胞凋亡机制探讨

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／LTFBP作用于Hep02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／LTFBP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／LTFBP作用于HepG-2细胞48h后凋亡相关基因Bcb2及Bax蛋白表达；比色法检测3、6、9和12g／LTFBP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HepG2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HepG2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Hepp2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

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

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.