MDR1及MDR3基因沉默逆转卵巢上皮性癌细胞对紫杉醇耐药的实验研究  

Reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel

作　　者：肖兰[1] 高瑞[1] 卢实[1] 卢美松[2] 梁铭霖[1] 任利容[1] 王泽华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院妇产科,武汉430022 [2]哈尔滨医科大学附属第一医院妇产科

出　　处：《中华妇产科杂志》2007年第6期412-416,共5页Chinese Journal of Obstetrics and Gynecology

摘　　要：目的研究多药耐药基因——MDR1及 MDR3基因沉默逆转卵巢上皮性癌(卵巢癌)细胞 A2780/taxol 对紫杉醇耐药的作用。方法用真核质粒介导的针对 MDR1及 MDR3基因的短发夹状 RNA(shRNA)转染 A2780/taxol 细胞(分别为 MDR1组、MDR3组),空质粒转染作为对照(空载体组)。流式细胞仪分别检测细胞早期凋亡、罗丹明123(Rh123)积聚情况,末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸标记法(TUNEL)检测细胞晚期凋亡情况,四甲基偶氮唑蓝(MTT)比色法检测细胞对紫杉醇的半数抑制浓度(IC_(50)),RT-PCR 技术检测 MDR1及 MDR3 mRNA 的表达,蛋白印迹法(western blot)检测半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白的表达。结果转染后,MDR1组及MDR3组 A2780/taxol 细胞的早期凋亡率分别达(20.21±0.56)%和(10.87±1.24)%。MDR1、MDR3组 A2780/taxol 细胞内的 Rh123平均荧光强度分别为116.6±8.1、98.4±3.8,显著高于空载体组的40.2±1.6,差异有统计学意义(P<0.05)。TUNEL 检测显示细胞发生了晚期凋亡。MDR1、MDR3组A2780/taxol 细胞对紫杉醇的 IC_(50)明显下降,分别与空载体组比较,差异均有统计学意义(P<0.05)。转染48 h 后,A2780/taxol 细胞中 MDR1和 MDR3 mRNA 的表达水平分别下降了(73.3±0.8)%和(51.6±0.4)%;MDR1、MDR3组细胞 caspase-3的表达量分别为(80.8±2.6)%、(72.0±4.7)%,均较空载体组增加。结论 MDR1及 MDR3基因沉默能恢复 A2780/taxol 细胞对紫杉醇的敏感性并诱导细胞凋亡,从而逆转 A2780/taxol 细胞对紫杉醇的耐药性。Objective To investigate the reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxeL Methods shRNA plasmid vector specifically targeting MDR1 and MDR3 genes was transfected into A2780/taxol cells. The early stage cell apoptosis and the effect of intracellular rhodamine 123 （Rh123） accumulation were detected by flow cytometry （FCM）. The late stage cell apoptosis rate was detected by terminal deoxynucleotidyl transferase （TdT）-mediated deoxyuridine triphosphate （dUTP） nick end labeling （TUNEL）. The 50% inhibition concentration （ IC50 ） of paclitaxel on A2780/taxol cells was determined by methyl thiazolyl tetrazolium （MTI＇） assay. MDRI and MDR3 mRNA were assessed by RT-PCR, and caspase-3 protein was detected by western blot. Results After treatment with MDR1 and MDR3 shRNA plasmid vector, early apoptosis rate of A2780/taxol cells was （20. 21 ±0.56）% and （10. 87 ± 1.24）%, respectively. MDR1 and MDR3 shRNA could increase cellular Rh123 accumulation （ 116. 6 ±8.1 and 98.4 ±3.8, respectively）. The late stage apoptosis rates detected by TUNEL displayed the same tendency as FCM results did. The IC50 for paclitaxel of A2780/taxol cells was decreased significantly. The mRNA levels of MDR1 and MDR3 in A2780/taxol cells were decreased by （73.3 ±0. 8） % and （51.6 ±0. 4）% of control, and the reduction of MDR1 and MDR3 mRNA was in a time-dependent manner. The expression of caspase-3 protein of MDR1 and MDR3 shRNA vector transfected group in A2780/taxol cells was significantly increased [（80.8 ± 2.6）% and （72.0 ± 4.7）%, respectively]. Conclusion MDR1 and MDR3 gene silencing could recover sensitivity of A2780/taxol cells to paclitaxel and induce cell apoptosis, thus reversing cell resistance to paclitaxel.

关 键 词：卵巢肿瘤 紫杉酚 基因 MDR RNA 小分子干扰 抗药性 肿瘤 

分 类 号：R737.31[医药卫生—肿瘤]