siA pollon抑制P-gp逆转白血病细胞耐药机制的研究

Study on the mechanism of siApollon inhibiting P-gp to reverse the drug resistance of leukemia cells

目的观察Apollon siRNA靶向沉默Apollon基因能否降低多药耐药(MDR)P糖蛋白(P-gp)表达,进而逆转K562/DNR多药耐药。方法将细胞分为实验组(脂质体转染靶向Apollon的siRNA K562/DNR)、细胞对照组(K562/DNR)和阴性对照组(脂质体转染的siRNAnc)。采用逆转录-聚合酶链式反应(RT-PCR)法检测三组细胞Apollon mRNA表达情况;采用Western Blotting法检测三组P-gp糖蛋白表达水平;采用四甲基偶氮唑盐(MTT)检测细胞转染前后对长春新碱(VCR)、柔红霉素(DNR)的敏感性。比较三组细胞Apollon mRNA表达情况、P-gp糖蛋白表达情况;分析细胞转染前后对VCR、DNR的敏感性。结果实验组Apollon mRNA的相对表达量为(24.233±0.108)%,低于细胞对照组的(90.031±0.182)%和阴性对照组的(83.686±0.076)%,差异有统计学意义(P<0.05)。实验组P-gp糖蛋白表达水平低于细胞对照组及阴性对照组,差异均有统计学意义(P<0.05)。实验组K562/DNR的VCR、DNR的IC50值明显高于细胞对照组和阴性对照组,差异具有统计学意义(P<0.05)。实验组未加药、DNR、VCR的K562/DNR的凋亡率均显著高于细胞对照组的(37.120±0.309)%和阴性对照组的(36.316±0.193)%,差异具有统计学意义(P<0.05);K562/DNR对化疗药VCR和DNR敏感性明显增强。结论siApollon能显著抑制K562/DNR细胞增殖,且P-gp的表达明显降低,提高K562/DNR细胞对化疗药物的敏感性,促进K562/DNR的凋亡,提示RNA干扰Apollon基因表达能在很大程度上提高K562/DNR对化疗药物的敏感性。

Objective To observe whether Apollon siRNA targeted silencing of Apollon can reduce the expression of multidrug resistance(MDR) P glycoprotein(P-gp), thereby reversing K562/DNR multidrug resistance. Methods The cells were divided into experimental group(liposome transfected siRNA K562/DNR targeting Apollon), cell control group(K562/DNR) and negative control group(liposome transfected siRNAnc). The expression of Apollon mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR), the expression of P-gp glycoprotein was detected by Western blotting, and the sensitivity to vincristine(VCR) and daunorubicin(DNR) before and after transfection was detected by methyl thiazolyl tetrazolium(MTT) in experimental group, cell control group, and negative control group. The expression of Apollon mRNA and P-gp glycoprotein in the three groups were compared, and the sensitivity to VCR and DNR before and after transfection was analyzed. Results The relative expression of Apollon mRNA in the experimental group was(24.233±0.108)%, which was lower than(90.031±0.182)% in the cell control group and(83.686±0.076)% in the negative control group. The difference was statistically significant(P<0.05). The expression level of P-gp glycoprotein in the experimental group was lower than that in the cell control group and the negative control group, and the difference was statistically significant(P<0.05). The VCR and DNR 50% inhibition concentration(IC50) of K562/DNR in the experimental group were significantly higher than those of the cell control group and the negative control group, and the difference was statistically significant(P<0.05). The apoptosis rate of K562/DNR in the experimental group without medicine, DNR and VCR was significantly higher than(37.120±0.309)% of the cell control group and(36.316±0.193)% the negative control group, and the difference was statistically significant(P<0.05);K562/DNR was significantly sensitive to the chemotherapy drugs VCR and DNR. Conclusion siApollon can significantly inhibit the proliferation of K562/DNR cells, and the expression of P-gp is significantly reduced, which improves the sensitivity of K562/DNR cells to chemotherapeutic drugs and promotes the apoptosis of K562/DNR, suggesting that RNA interference with Apollon gene expression can greatly improve the sensitivity of K562/DNR to chemotherapy drugs.