bcl-2-反义寡核苷酸对卵巢癌顺铂耐药细胞株的耐药逆转作用

bcl-2 antisense oligodeoxynucleotides on reversal ■ cisplatin resistance in ovarian carcinoma cell line A2780/DDP

目的 探讨bcl-2-反义寡核苷酸(bcl-2-AODN)对卵巢癌顺铂(DDP)耐药细胞耐药性的逆转作用。方法 以卵巢癌顺铂耐药细胞株A2780/DDP为模型,用脂质体转染的方法,将bcl-2-AODN转染A2780/DDP细胞,同时以转染bcl-2正义链(bcl-2-SODN)作对照。采用DNA凝胶电泳检测转染后是否诱导细胞出现凋亡,应用免疫组织化学技术检测Bcl-2蛋白表达变化,RT-PCR检测Bcl-2mRNA表达水平的变化。MTT法检测细胞抑制率与药物半数抑制浓度。结果 脂质体转染bcl-2反义寡核苷酸12 h后,倒置显微镜下即可见A2780/DDP有40％细胞出现变化,收集细胞检测,Bcl-2蛋白表达降低;实验组bcl-2mRNA表达水平与转染前相比,基因扩增条带亮度明显减弱,mRNA表达率下降(P<0.05);DNA凝胶电泳出现梯带状条带;细胞药物半数抑制浓度下降(P<0.05)。结论 bcl-2-AODN可通过增加细胞的凋亡而一定程度地逆转卵巢癌细胞的顺铂耐药性,提高卵巢癌细胞对化学治疗药物的敏感性。

Objective To evaluate the influence of bcl-2 antisense oligodeoxynucleotides (AODN) on reversal of cisplatin resistance in ovarian cancer cells. Methods Cisplatin reistance ovarian carcinoma cells were served as models. The bcl-2 antisense oligodeoxynucleotides were transfected to the cells by lipofectamine and the sense-oligodeoxyuncleotides were used as control. The level of bcl-2 mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The apoptosis of ovarian cancer cell lines were measured by DNA ladder assay. Protein was measured by immunofluorescence technique. The IC_(50) was measured by MTT. Results After 12 h of transfection by lipofectamine, it was seen that 40% cells were changed. We could not differentiate whether it was apoptosis or necrosis by the inverted phase contrast microscope, but we could observe DNA 'ladder' with agrose gel electrophoresis in the antisense-transfected cells. PT-PCR showed the expression level of bcl-2 mRNA in the transfected cells was significantly lower than before and the expression of Bcl-2 protein was decreased, the IC_(50) was deceased. Conclusion bcl-2-AODN can reverse the resistance to cisplatin in ovarian cancer cell line A2780/DDP through inhibiting the expression of Bcl-2 protein and increasing the apoptosis.