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目的探讨应用RNA干扰(RNAi)技术逆转卵巢上皮性癌(卵巢癌)细胞多药耐药的可行性。方法将设计合成的针对多药耐药基因MDR1的特异性小分子干扰RNA(siRNA),用脂质体转染具有MDR1基因高表达的卵巢癌紫杉醇耐药细胞株OVCAR8/TR。用荧光定量RT-PCR技术和流式细胞仪分别测定转染前后细胞MDR1mRNA及糖蛋白P-gp表达的变化;三磷酸腺苷(ATP)生物荧光法检测转染前后细胞对顺铂、氟尿嘧啶、阿霉素和紫杉醇药物敏感性(以ATP抑制率判断)的变化情况。结果转染后24、48、72、96和120h,OVCAR8/TR细胞MDR1mRNA的抑制率分别为26·42%、84·00%、78·43%、45·85%和0;转染后48h MDR1mRNA的抑制率达到最高峰。转染后24、48、72、96和120h,OVCAR8/TR细胞P-gp的抑制率分别为16·71%、49·64%、85·23%、65·98%和9·44%;转染后72h P-gp的抑制率达到最高。转染MDR1siRNA后,能够明显提高OVCAR8/TR细胞对紫杉醇和阿霉素的敏感性,在紫杉醇的作用下,转染前后OVCAR8/TR细胞的ATP抑制率分别为(25·8±3·1)%和(78·0±9·8)%,转染前后比较,差异有统计学意义(P<0·05)。结论在OVCAR8/TR细胞中,针对MDR1合成的siRNA能够有效地抑制MDR1mRNA和P-gp的表达,并能恢复其对紫杉醇和阿霉素的敏感性。应用RNAi技术,能够逆转卵巢癌细胞对化疗药物的多药耐药。
Objective To investigate the feasibility of reversing multidrug resistance in epithelial ovarian cancer (ovarian cancer) cells by RNA interference (RNAi) technique. Methods Specific small interfering RNA (siRNA) targeting multidrug resistance gene MDR1 was designed and synthesized. Ovarian cancer cell line OVCAR8 / TR with overexpression of MDR1 gene was transfected with liposome. The changes of MDR1mRNA and P-glycoprotein P-gp expression in cells before and after transfection were detected by real-time fluorescence quantitative RT-PCR and flow cytometry. The changes of MDR1mRNA, And paclitaxel drug sensitivity (to determine the rate of ATP inhibition) changes. Results The inhibitory rates of MDR1 mRNA in OVCAR8 / TR cells were 26.42%, 84.00%, 78.43%, 45.85% and 0 at 24, 48, 72, 96 and 120 h after transfection, respectively. MDR1 mRNA inhibition reached its peak. The inhibitory rates of P-gp in OVCAR8 / TR cells were 16.71%, 49.64%, 85.23%, 65.98% and 9.44% at 24, 48, 72, 96 and 120 h after transfection respectively. The inhibition rate of P-gp reached the highest at 72h after transfection. The transfection of MDR1siRNA significantly increased the sensitivity of OVCAR8 / TR cells to paclitaxel and doxorubicin. Under the action of paclitaxel, the inhibitory rates of ATP in OVCAR8 / TR cells before and after transfection were (25.8 ± 3.1) % And (78 · 0 ± 9 · 8)% respectively. There was a significant difference between before and after transfection (P <0.05). Conclusions siRNA targeting MDR1 can effectively inhibit the expression of MDR1 mRNA and P-gp and restore its sensitivity to paclitaxel and doxorubicin in OVCAR8 / TR cells. The use of RNAi technology can reverse the multidrug resistance of ovarian cancer cells to chemotherapeutic drugs.