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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 study the effect of MDR1 and MDR3 gene silencing on paclitaxel resistance in A2780 / taxol ovarian cancer cells. Methods A2780 / taxol cells (MDR1 and MDR3, respectively) were transfected with eukaryotic plasmids containing short hairpin RNA (shRNA) targeting MDR1 and MDR3 genes and empty plasmid transfected as control (empty vector group). Flow cytometry was used to detect the early apoptosis of cells, the accumulation of rhodamine 123 (Rh123), the late apoptosis of cells by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate labeling (TUNEL) The half inhibitory concentration of paclitaxel (IC 50) was determined by MTT assay. The expression of MDR1 and MDR3 mRNA was detected by RT-PCR. The cysteine day was detected by western blot Aspartate proteinase 3 (caspase-3) protein expression. Results After transfection, the early apoptotic rates of A2780 / taxol cells in MDR1 group and MDR3 group were (20.21 ± 0.56)% and (10.87 ± 1.24)%, respectively. The mean fluorescence intensity of Rh123 in A2780 / taxol cells in MDR1 and MDR3 groups was 116.6 ± 8.1 and 98.4 ± 3.8, respectively, which was significantly higher than that in empty vector group (40.2 ± 1.6) (P <0.05). TUNEL assay showed late apoptosis occurred in cells. The IC 50 of paclitaxel in A2780 / taxol cells in MDR1 and MDR3 groups were significantly decreased compared with those in empty vector group (P <0.05). The expression of MDR1 and MDR3 mRNA in A2780 / taxol cells decreased by (73.3 ± 0.8)% and (51.6 ± 0.4)%, respectively, and the expression levels of caspase-3 in MDR1 and MDR3 cells were (80.8 ± 2.6%, and (72.0 ± 4.7)%, respectively. Conclusion Silencing MDR1 and MDR3 can restore the sensitivity of A2780 / taxol cells to paclitaxel and induce apoptosis, thereby reversing the drug resistance of A2780 / taxol cells to paclitaxel.