黄芪甲苷逆转人肝癌HepG2／GCS细胞多药耐药的作用及其机制  

The function and mechanism of astragaloside IV on the chemoresistance of HepG2/GCs cell lines

作　　者：田彦璋[1] 高飞[1] 赵海潮 赵浩亮[1] Tian Yanzhang;Gao Fei;Zhao Haichao;Zhao Haoliang(Department of General Surgery,Shanxi Dayi Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医学科学院山西大医院普通外科,太原030001 [2]山西医科大学研究生院

出　　处：《中华肝胆外科杂志》2018年第8期555-559,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：山西省自然科学基金（2014012）

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（Westernblot）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Hoechst33258染色。流式细胞术检测各组细胞凋亡率，Westernblot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Westernblot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10μg/ml、10μg/ml、15μg/ml。Hoechst33258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。Objective To investigate whether astragaloside IV can regulate the muhidrug resistance of HepG2/GCS-resistant cell lines, restore the sensitivity of drug-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. Ho- echst 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 observa- tion 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 cyto-toxicity 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 ceils 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 multi- drug 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.

关 键 词：癌 肝细胞 多药耐药 黄芪甲苷 阿霉素 

分 类 号：R285[医药卫生—中药学]