摘要目的 探讨黄芪甲苷(AST)逆转HepG2/GCS细胞的多药耐药及其机制.方法 用重组葡萄糖神经酰胺合成酶(GCS)质粒及空质粒转染HepG2肝癌细胞,反转录聚合酶链反应(RT-PCR)和蛋白印迹(Western blot)分析GCS以及多药耐药(MDR)基因的表达.GCS转染组、空白载体组和空白对照组行AST细胞毒性实验和阿霉素(ADM)体外肿瘤细胞抑制实验,GCS转染组药物干预后行Hoechst 33258染色.流式细胞术检测各组细胞凋亡率,Western blot检测各组Caspase-9、Caspase-3、Bax、Bcl-2表达.结果 荧光显微镜观察到GCS转染高表达.RT-PCR以及Western blot显示,GCS转染组GCS和MDR1的相对表达明显高于空白载体组,差异有统计学意义(P＜0.05).AST对各组细胞无明显毒性.空白对照组、空白载体组和GCS转染组ADM的50％细胞抑制浓度分别为10 μg/ml、10 μg/ml、15μg/ml.Hoechst 33258和流式细胞术检测表明,GCS转染组、ADM+GCS转染组、ADM+AST+GCS转染组凋亡率呈升高趋势,凋亡率分别为(2.68±0.20)％比(32.66±1.84)％和(32.66±1.84)％比(64.29±1.94)％,差异有统计学意义(P＜0.05).ADM+ AST+ GCS转染组的Caspase-9、Caspase-3、Bax相对表达均高于ADM+GCS转染组,Bcl-2相对表达低于ADM+GCS转染组,差异有统计学意义(P＜0.05).结论 AST可逆转HepG2/GCS细胞多药耐药,增强其对ADM的敏感性,通过Caspase途径和Bax途径促进肿瘤细胞凋亡.

abstractsObjective To investigate whether astragaloside Ⅳ can regulate the multidrug resistance of HepG2/GCS-resistant cell lines,restore the sensitivity of drag-resistant cell lines to adriamycin (ADM) and its mechanism.Methods We used recombinant GCS (shRNAS) and control recombinant plasmids and did the transfection with HepG2 cells.RT-PCR and Western blot were used to analyze the expression of GCS.Astragaloside Ⅳ cytotoxicity experiments and ADM were performed in experimental and control groups.Hoechst 33258 was detected in two groups,apoptosis was detected by flow cytometry,and protein expression of caspase 9,3,Bax,and bcl-2 were detected by Western blot.Results RT-PCR and fluorescence observation showed that GCS was highly expressed after the transfection.Western blot showed that compared with control group,and HepG2GCS group,GCS and MDR1 expression were higher than;Astragaloside Ⅳ cytotoxicity experiment showed tumor proliferation was not regulated by GCS(FHepG2GCS=0.308,FHepG2EV =0.216,FHepG2 =0.153,P＞ 0.05),ADM in vitro tumor cell inhibition experiments showed that HepG2GCS cells were resistant to ADM (50％ cell transplantation concentration were 7.5,7.5,15 μg/ml);Hoechst 33258 and flow cytometry showed that Astragaloside Ⅳ can restore ADM tumor inhibition;Western blot showed that compared to untreated HepG2EV and HepG2GCS cells,protein level of caspase 9,caspase 3 were increased in Ast+HepG2EV and Ast+HepG2GCS groups (t=7.17,P＜0.05).At the same time,Bax and Bcl-2 were significantly different in each group (P＜ 0.05).Conclusions Astragaloside Ⅳ reverses multidrug resistance in HepG2/GCS cell lines,restores its sensitivity to ADM,promotes apoptosis in tumor cells through the caspase pathway and Bax pathway,and thus plays an important role in cancer chemotherapy.