人参皂苷Rh2对人肝癌细胞HepG2/ADM耐药逆转作用及其机制研究  

Study on the drug-resistant reversal effects of ginsenoside Rh2 in human hepatocellular carcinoma HepG2/ADM cells and its mechanism

作　　者：吴艳林[1] 刘润田[2] 

机构地区：[1]南阳市中心医院肿瘤科,南阳医学硕士473000 [2]河北医科大学第二医院肝胆外科,石家庄050000

出　　处：《医学研究生学报》2017年第5期476-480,共5页Journal of Medical Postgraduates

基　　金：河北省医学科学研究重点课题计划指令项目(20150227)

摘　　要：目的人参皂苷Rh2能抑制多种恶性肿瘤细胞增殖,但缺少Rh2对多药耐药的肝癌细胞的敏感性研究。文中主要探讨人参皂苷Rh2对人肝癌细胞株HepG2/ADM多药耐药性[盐酸多柔比星(ADM)、5-氟尿嘧啶(5-Fu)、顺铂(DDP)、长春新碱(VCR)]的逆转作用及机制。方法 MTT法检测(0~250μg/m L)Rh2对HepG2/ADM细胞活力的影响;MTT法筛选最佳逆转耐药的Rh2浓度。细胞分为空白对照组、ADM组和ADM+40μg/m LRh2组。空白对照组:不加药物处理;ADM组:给予ADM处理48 h;ADM+40μg/m LRh2组:给予40μg/m L的Rh2预处理30 min后,给予ADM处理48 h。流式细胞术检测Rh2对细胞内Rh-123荧光强度的影响;RT-PCR法检测MDR1基因的表达;Western blot检测P-gp、Bax、Bcl-2、cleved caspase-3蛋白水平。结果与HepG2细胞相比,HepG2/ADM对ADM、DDP、5-FU、VCR 4种化疗药物的耐药指数分别为32.95、4.63、4.20、4.81。经过40μg/m L的Rh2作用HepG2/ADM细胞48 h后,耐药细胞对4种化疗药的敏感性增强且IC50明显下降,其耐药性的逆转倍数分别为3.70、3.53、2.64、2.55倍。耐药细胞内储留的Rh-123的荧光强度通过流式细胞仪检测结果显示,与ADM组比较,加入40μg/m L Rh2后,细胞内Rh-123的荧光强度明显增高(65.83±1.78 vs 78.21±1.26,P<0.01)。RT-PCR结果显示,ADM+40μg/m L Rh2组MDR1表达较ADM组显著降低(0.48±0.02 vs 0.86±0.05,P<0.05)。Western blot结果显示,ADM+40μg/m L Rh2组P-gp蛋白水平亦较ADM组明显降低(0.97±0.04 vs 1.91±0.03,P<0.01);ADM+40μg/m L Rh2组Bax和cleaved caspase-3表达较ADM组明显增加(1.76±0.04 vs 1.25±0.02,38.26±5.45 vs 0.42±0.04,P<0.05),同时发现Bcl-2表达明显减少(1.25±0.05 vs 1.86±0.03,P<0.05)。结论人参皂苷Rh2能有效逆转HepG2/ADM细胞的多药耐药性,其作用机制可能与降低MDR1、P-gp表达、增加细胞内药物积累以及介导Bax/Bcl-2信号通路有关。Objective Ginsenoside Rh2 can inhibit the proliferation of a variety of malignant tumor cells. However,little research has been done on the sensitivity of Rh2 in human hepatocellular carcinoma HepG2/ADM cells with multidrug resistance（ MDR）.This study aimed to explore the reversing effects of ginsenoside Rh2 on the MDR of human hepatocellular carcinoma HepG2/ADM cells and its potential mechanism. Methods MTT assay was applied to detect the effect of Rh2（ 0-250 μg/m L） on the viability of HepG2/ADM cells and screen out the optimum drug-resistant reversal concentration of Rh2. Cells were divided into 3 groups： HepG2/ADM group（ without any medicine treatment）,ADM group（ ADM treatment for 48 h）,ADM＋40 μg/m L Rh2 group（ pretreatment of 40μg/m L Rh2 for 30 min followed by ADM treatment for 48 h）. Flow cytometry was applied to detect the effect of Rh2 on the fluorescence intensity of cellular Rh-123. RT-PCR was used to measure the expression of MDR1 gene. Western blot was used to detect the protein levels of P-gp,Bax,Bcl-2 and cleaved caspase-3. Results 40 μg/m L ginsenoside Rh2 significantly reversed the MDR of HepG2/ADM cells by a 2.55-to-3.70-fold increase in sensitivity. Furthermore,compared with ADM group,the efflux of Rh-123 in HepG2/ADM cells were remarkably inhibited by Rh2 in ADM＋40 μg/m L Rh2 group（ 65.83±1.78 vs 78.21± 1.26,P〈 0.01）,along with the down-regulated expressions of MDR1（ 0.48±0.02 vs 0.86±0.05,P〈 0.05）,P-gp（ 0.97± 0.04 vs 1.91± 0.03,P〈 0.01）,Bcl-2（ 1.25±0.05 vs 1.86±0.03,P〈0.05） and the up-regulated protein level of Bax（ 1. 76 ± 0. 04 vs 1. 25 ± 0. 02,P 〈 0. 05） and cleved caspase-3（ 0.42±0.04 vs 38.26±5.45,P〈0.05）. Conclusion Ginsenoside Rh2 can effectively reverse the MDR of HepG2/ADM cells,and the potential mechanism is related to the decreased expressions of MDR1 and P-gp,the increasing drug concentration inside the cells and the Bax/Bcl-2 signaling pathway.

关 键 词：人参皂苷RH2 HepG2/ADM细胞 多药耐药性 MDR1/P-GP BAX/BCL-2 

分 类 号：R735.7[医药卫生—肿瘤]