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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。
AIM: To investigate the mechanism of total flavonoids of Broussonetia papyrifera (TFBP) inducing apoptosis in HepG-2 hepatocellular carcinoma cells. Methods: MTT assay was used to detect the effects of 3, 6, 9 and 12 g / L TFBP on the growth of HepG-2 cells at 24, 48 and 72 hours respectively. Hoechst33342 fluorescence staining was used to observe the effects of 3, 6, 9 and 12 g / L TFBP in HepG-2 cells for 48h, the expressions of Bcl-2 and Bax protein in HepG-2 cells were detected by immunocytochemistry after treated with 3, 6, 9 and 12g / L TFBP for 48h; The activity of Caspase-3 in HepG-2 cells treated with 3, 6, 9 and 12 g / L TFBP for 48 h was detected by colorimetric assay. Results: With the increase of drug concentration, the effect of TFBP on proliferation of HepG-2 cells gradually increased in a dose-dependent manner. After treated with 3, 6, 9 and 12 g / L TFBP for 24 h, HepG- The inhibitory rates of cell proliferation were 7.63%, 11.86%, 19.02% and 21.81%, F = 5.16, P = 0.034, respectively. The inhibitory rates were 20.20%, 29.44%, 39.21% and 43.96% ; 27.24%, 34.70%, 52.46% and 64.72% respectively after 72h, F = 86.78, P <0.001. Hoechst33342 fluorescence staining showed that, with the increase of drug concentration, the growth of cells slowed down, the cytoplasm became loose, the nucleus was shrunken, and the typical apoptotic morphology appeared. Immunocytochemistry showed that the expression of Bcl-2 protein in HepG-2 cells gradually became lighter with the increase of concentration of HepG-2 cells treated with different concentrations of TFBP for 48h, and the protein expression of Bax gradually became deeper with the increase of concentration. The positive rate of Bcl and Bax protein expression was significantly different from the control group, with F values of 6.563 and 30.883, P values of 0.012 and 0.001, respectively. Caspase-3 activity assayed by colorimetric assay showed that Caspase-3 activity of HepG-2 cells treated with 3, 6, 9 and 12 g / L TFBP for 48 h was (1.38 ± 2.13), (3.58 ± 2.21 ), (4.95 ± 3.61), (5.25 ± 1.41) and (5.75 ± 2.24) U / mg respectively. With the increase of TFBP concentration, the activity of Caspase-3 was significantly increased. When TFBP concentrations were 6, 9 and 12 g / L , The experimental group and the control group, the difference was statistically significant, F = 5.42, P = 0.003. Conclusion: TFBP can inhibit HepG-2 hepatoma cell proliferation and induce apoptosis in vitro. Its mechanism of apoptosis may be related to its down-regulation of Bcl-2 protein and up-regulation of Bax protein expression and enhancement of Caspase-3 activity.