β-榄香烯注射液对人肝癌HepG2细胞微管系统的影响

Effects of β-elemene on Microtubule System of Human Hepatocarcinoma HepG2 Cells

目的:探讨β-榄香烯对人肝癌细胞HepG2生长抑制作用及微管系统的影响。方法:将实验分为对照组(未加药物)与实验组(不同浓度β-榄香烯注射液0.02,0.04,0.08 g.L-1组);采用四甲基偶氮唑蓝(MTT)法测定β-榄香烯对HepG2细胞增殖的影响,流式细胞技术分析肿瘤细胞周期时相分布,激光共聚焦显微技术观察β-榄香烯处理的HepG2细胞内微管的表达、分布;逆转录-多聚酶连反应技术(RT-PCR)观察微管蛋白βmRNA水平表达;采用蛋白免疫印迹法(Western-blot)分析微管蛋白β蛋白水平的表达、聚合和未聚合微管蛋白含量的变化。结果:0.1,0.08,0.06,0.04,0.02,0.01 g.L-1的β-榄香烯注射液对HepG2细胞增殖具有抑制作用,抑制效应呈时间和浓度依赖性;将细胞阻滞在S期。激光共聚焦分析表明,β-榄香烯不同浓度作用的HepG2细胞微管网络异常;β-榄香烯注射液抑制HepG2细胞β微管蛋白mRNA表达,呈浓度依赖性。蛋白免疫印迹显示β-榄香烯能抑制微管蛋白β的表达,同时抑制细胞内微管蛋白的聚合,呈浓度依赖性。结论:β-榄香烯注射液能够抑制人肝癌细胞HepG2增殖,下调微管蛋白β的表达,抑制HepG2细胞内微管的聚合可能是造成HepG2细胞生长抑制的因素之一。

Objective： To investigate the effect of β-elmene on the growth of human hepatocarcinoma HepG2 cells and the effect on microtubular. Method： Proliferation of HepG2 cells was evaluated by MTT assay. Cell cycle of HepG2 cells was analyzed by flow cytometry.The expression and distribution of microtubule in HepG2 cells were investigated by confocal microscopy. The mRNA expression of β-tubulin was detected by RT-PCR. Immunoblotting analysis was used to determine protein expression of β-tubulin and the proportion of polymerization of tubulin. Result： β-elmene inhibited the proliferation of HepG2 cells in a dose-dependent and time-dependent manner, and induce tumor arrested cells at S-phase.Confocal microscopy showed an abnormal microtubular network in HepG2 cell treated with β-elmene. RT-PCR and Western-blot analysis showed that β-elmene down-regulated β-tublin at both mRNA and protein level. The mRNA expression of β-tubulin in the observed groups were lower than that in the control group and presented a dose-dependent manner. Further analysis by immunoblotting confirmed the down-regulation of β-elmene on the expression for β-tubulin in a dose-dependent manner.Moreover, β-elmene reduced the proportion of polymerization of microtubule in a dose-dependent manner. Conclusion： β-elmene could inhibit the proliferation of HepG2 cells,down-regulation of the expression of β-tubulin and inhibit the microtubular polymerization might be one of mechanism that β-elmene contribute to growth inhibition of HepG2 cell.