不同粒径及纯度的雄黄颗粒对HepG2细胞的凋亡诱导作用

Effects of realgar particles with different particle sizes and purity on apoptosis of HepG2 cells

目的 探讨不同粒径及纯度的雄黄颗粒对HepG2细胞的凋亡诱导作用及其作用机制.方法 按前期实验方法制备各组不同粒径及纯度的雄黄颗粒,实验分为6组：天然雄黄（CR）组、纯化雄黄（PR）组、天然纳米雄黄（CRN）组、纯化纳米雄黄（PRN）组、丝裂霉素（Mit）组、对照组,分别使用相应药物处理HepG2细胞,对照组不进行处理.噻唑蓝（MTT）法检测不同浓度的各组雄黄颗粒作用HepG2细胞12、24、48、72 h后的细胞增殖率并筛选最佳作用浓度和作用时间.各组HepG2细胞分别经20.0 μ/ml的不同药物处理24 h后观察细胞形态学变化,流式细胞术测定凋亡率,实时PCR测定Bcl-2、BaxmRNA表达水平.结果 MTT法显示,经不同组别雄黄颗粒处理后HepG2细胞增殖率均有不程度降低,且其抑制作用具有时间效应和浓度效应;通过MTT法筛选出最佳作用浓度及作用时间为20.0 μ/ml和24 h.细胞形态学检测发现各组雄黄颗粒能诱导HepG2细胞出现核溶解、破裂等特征性凋亡形态变化.流式细胞术证实各组雄黄颗粒均可诱导HepG2细胞发生凋亡,主要表现为早期凋亡.实时PCR显示,与对照组比较,不同组别雄黄颗粒处理后Bcl-2 mRNA表达下调,Bax mRNA表达上调（均P＜0.05）.结论 雄黄颗粒对HepG2细胞的诱导凋亡作用随着粒径的降低及纯度的提高而增强,其作用机制可能与下调Bcl-2基因表达、上调Bax基因表达有关.

Objective To explore the apoptosis-inducing effect of realgar particles with different particle sizes and purity on HepG2 cells and the underlying mechanism.Methods Realgar particles with different particle sizes and purity were prepared according to methods in previous experiments.In the present study,6 groups were set,including the crude realgar （CR） group,purified realgar （PR） group,crude reaglar nanoparticles （CRN） group,purified realgar nanoparticles （PRN） group,mitomycin （Mit） group and the control group.HepG2 cells in these groups were treated with the corresponding agent,except in the control group.MTT method was used to determine the proliferation rate of HepG2 cells at 12,24,48 and 72 h,and thereby the optimal treatment concentration and duration were evaluated.After HepG2 cells were treated by 20.0 μg/ml realgar particles in each group for 24 hours,the cell morphology was examined;apoptosis rate was determined by flow cytometry.Real-time PCR was used to determine the Bcl-2 and Bax mRNA expression.Results The MTT results showed the proliferation rate in all treatment groups of HepG2 cell was obviously reduced in a time-and concentration-dependent manner.According to MTT method,treatment for 24 h at 20.0 μg/ml was determined as the optimal acting time and concentration.Cell morphology revealed characteristic features of aptopsis such as lysis and rupture of HepG2 cell nuclei as induced by realgar particles in each treatment group.Flow cytometry confirmed that the realgar particles in treatment groups can induce HepG2 cells apoptosis,especially during the early phase of cell growth.Realtime PCR revealed the down-regulation of Bcl-2 mRNA and up-regulation of Bax mRNA in the realgar particles treated groups compared with those in the control group （all P〈0.05）.Conclusion Different realgar particles can induce HepG2 apoptosis.The apoptosis-inducing effect becomes stronger with smaller particle sizes and higher purity.The underlying mechanism may be related with the down-regulation of Bcl-2 and up-regulation of Bax.