E2F-1基因沉默对人胃癌耐药细胞株SGC7901／顺铂多药耐药性的逆转  

Reversion of multidrug resistance of human gastric cancer SGC7901/DDP cells by E2F-1 gene silencing

作　　者：廉超[1] 杨杰[1] 王晓通[1] 谢玉波[2] 肖强[1] 

机构地区：[1]广西医科大学第一附属医院胃肠腺体外科,南宁530021 [2]广西医科大学第一附属医院麻醉科,南宁530021

出　　处：《中华肿瘤杂志》2014年第3期171-176,共6页Chinese Journal of Oncology

基　　金：国家自然科学基金（30860273）;广西自然科学基金（2013GXNSFAA019163）

摘　　要：目的探讨E2F一1基因沉默后对人胃癌顺铂（DDP）耐药细胞株SGC7901／DDP多药耐药性的影响及可能的作用机制。方法将人胃癌耐药细胞株SGC7901／DDP接种于6孔板，并分为以E2F-1基因沉默重组慢病毒颗粒Lv-shRNA—E2F-1感染的SGC7901／DDP细胞（实验组）、以对照慢病毒颗粒Lv-shRNA，NC感染的SGC7901／DDP细胞（阴性对照组）以及常规培养的SGC7901／DDP细胞（空白对照组）。采用Westernblot技术检测各组细胞中E2F-1蛋白的表达，采用四甲基偶氮唑蓝法检测阿霉素、氟尿嘧啶和DDP对各组细胞的半数抑制浓度（IC50），采用流式细胞术检测各组细胞中阿霉素的泵出率和细胞凋亡率，采用半定量RT—PCR和Westernblot技术检测各组细胞中多药耐药基因1（MDRl）、多药耐药相关蛋白（MRP）及凋亡相关基因cyclinD1、c-Myc、Skp2mRNA和蛋白的表达水平。结果实验组、阴性对照组和空白对照组SGC7901／DDP细胞中E2F-1蛋白的相对表达量分别为0．794±0．033、1．487±0．082和1．5114-0．084，实验组SGC7901／DDP细胞中E2F-1蛋白的相对表达量较阴性对照组和空白对照组分别下降46．6％和47．5％（P〈0．01）。阿霉素、氟尿嘧啶和DDP对实验组SGC7901／DDP细胞的Ic50均较阴性对照组和空白对照组明显降低（均P〈0．01）。实验组、阴性对照组和空白对照组SGC7901／DDP细胞中阿霉素的泵出率分别为（0．16±0．01）％、（0．37±0．01）％和（0．35±0．02）％，实验组SGC7901／DDP细胞中阿霉素的泵出率明显低于阴性对照组和窄白对照组（P〈0．01）。实验组、阴性对照组和空白对照组SGC7901／DDP细胞的凋亡率分别为（33．82±1．26）％、（17．34±0．81）％和（13．164-1．06）％，实验组SGC7901／DDP细胞的凋亡率明显高于阴性对照组和空白对照组（P〈0．01）。与阴性对照组和空白对照组比较，实验组SGC7901／DDP细胞中MDRl、MRP、cyclObjective To investigate the effects of E2F-1 gene silencing on multidrug resistance of human gastric cancer SGC7901/DDP cells and its possible mechanisms. Methods Gastric cancer SGC7901/DDP cells were seeded in 6 well plates and divided into three groups： the experimental group, blank control and the negative control groups. For the experimental group, the SGC7901/DDP ceils were transfected with recombinant lentivirus vector （ Lv-shRNA-E2F-1 ）, while the negative control with an control lentiviral vector （Lv-shRNA-NC） and the blank control with no treatment. The E2F-1 protein level was analyzed by Western blot. MTT assay was used to detect the half maximal inhibitory concentration （IC50） of three chemotherapy drugs including adriamycin, 5-fluorouracil （5-Fu） and cisplatine （DDP） of the three cell groups. Flow cytometry （FCM） was used to detect the pump-out rate of adriamycin and apoptosis rate of the three cell groups. Semi-quantitative RT-PCR and Western blot were also used to detect the protein and mRNA levels of muhidrug resistance-associated genes （ MDR1, MRP） and apoptosis-related genes （ c-Myc, Skp2, cyclinD1 ）. Results The expression of E2F-I protein in the experimental group was significantly lower than that in the negative control and blank control groups （0. 794 ± 0. 033 vs. 1. 487 ± 0. 082 vs. 1.511 ± 0. 084, P 〈 0.01 ）. The IC50 of the three chemotherapy drugs （ adriamycin, 5-Fu and cisplatine ） in the experimental group was significantly lower than that of the negative control and blank control groups, respectively （P 〈 0.01 ）. Compared with the negative control and blank control groups, the pump-out rate of adriamycin of the experimental group was significantly declined [ （0.16 _＋ 0.01 ） % vs. （0.37 _＋ 0.01 ） % vs. （0.35 ＋0.02）%, P 〈 0.01 ]. However, the apoptosis rate of the experimental group was significantly higher than that of the negative control and blank control groups [ （ 33.82 ^- 1.26） % vs. （ 17.34 ＋ 0.81 �

关 键 词：E2F-1基因 胃肿瘤 抗药性 肿瘤 顺铂 

分 类 号：R735.2[医药卫生—肿瘤]