槐耳清膏诱导人直肠癌HR8348细胞凋亡的实验研究

Study on Apoptosis of Human Rectal Adenocarcinoma Cell Line HR8348 Induced by Polysaccharide of Trametes Robiniophila Murr in Vitro

目的 探讨槐耳清膏在体外对人直肠癌HR83 48细胞的生长抑制作用和凋亡诱导作用及其作用机理。方法 将处于对数生长期的HR83 48细胞用含槐耳清膏的培养基培养 3 6h ,用四氮唑蓝 (MTT )比色法检测吸光度 (OD)值 ,计算抑制率 ;用甲基绿 派若宁染色法及TUNEL法检测细胞凋亡指数 ;用免疫组织化学方法检测bcl 2、bcl xl、bax、bak及 p5 3基因表达的情况。 结果 槐耳清膏对HR83 48细胞的抑制率随浓度增加而上升 ,浓度为 4.0mg/ml时抑制率最大 (71.1% ) ,与 5 FU组 (浓度为 10 μg/ml)相比差异无显著性意义 (P>0 .0 5 )。甲基绿 派若宁染色法和TUNEL法均可见到典型的细胞凋亡、凋亡小体或出泡现象 ,凋亡指数随槐耳清膏浓度的增加而增加 ,当浓度达 4.0mg/ml时 ,凋亡指数迅速上升 ,甲基绿 派若宁法为 0 .162 0± 0 .0 12 8,TUNEL法为 0 .2 612± 0 .0 15 8,均大于 5 FU组的凋亡指数(0 .0 780± 0 .0 0 95和 0 .10 2 8± 0 .0 13 1) ,P<0 .0 5。槐耳清膏组bcl 2、bcl xl、bak、p5 3蛋白表达较空白对照组明显增强(P<0 .0 5 ) ,而bax变化不明显 (P>0 .0 5 )。槐耳清膏组bak/bcl 2和bak/bcl xl比值显著大于空白对照组。结论 槐耳清膏在体外对人直肠癌HR83 48细胞具有显著的抑制作用和凋亡诱导作用。槐耳清膏?

Objective To investigate the growth inhibition and the apoptosis inducement effects of polysaccharide of trametes robiniophila murr (PS-T) on human rectal cancer cell line HR8348 and elucidate the possible mechanisms. Methods After treatment with PS-T, the growth inhibition rate of human rectal cancer cell strain HR8348 was studied by MTT method. The apoptotic index was detected by methyl green and pyronine Y staining and by terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling (TUNEL). The bcl-2, bcl-xl, bax, bak and p53 gene expressions of HR8348 cells were examined by immunohistochemical method. Results PS-T induced a dose-dependent inhibition of HR8348 cells in vitro . The maximum percentage of growth inhibition was 71.1 % by 36 h after administration of PS-T at a concentration of 4.0 mg/ml. There was no significant difference in the inhibitory rate as compared to the positive control (5-FU 10 μg/ml, P > 0.05). The typical apoptosis phenomenons were observed under the light microscope by both staining methods. The apoptotic index increased parallelling with the increase of concentration of PS-T. When the PS-T concentration was 4.0 mg/ml, the apoptotic index determined by methyl green and pyronine Y staining and by TUNEL method increased rapidly to 0.162 0 ±0.012 8 and 0.261 2±0.015 8, respectively, which was greater than that of the positive control (5-FU 10 μg/ml, P < 0.05). The expression of bcl-2, bcl-xl, bak and p53 was increased in the PS-T-treated HR8348 cells by 36 h, while the bax remained unchanged. Expression index of bak/bcl-2 and bak/bcl-xl was increased significantly as compared with the control ( P < 0.05). Conclusion PS-T can significantly inhibit growth and induce apoptosis of human rectal cancer cell strain HR8348 in vitro . The apoptosis induced by PS-T might be related to the increase of the ratio of bak to bcl-2 and to bcl-xl and upregulation of the expression of p53 gene.