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目的研究小干扰 RNA(siRNA)对白血病多药耐药细胞系 K562/ADM 细胞 mdr1基因表达的沉默作用和凋亡抑制的逆转效应。方法 K562/ADM 为靶细胞,设汁、筛选和合成2对针对mdr1基因 mRNA 的 siRNA(mdr1 siRNA-1和 mdr1 siRNA-2),用脂质体介导转染 K562/ADM 细胞;实时荧光定量 PCR(real-tMe PCR)法检测 mdr1 mRNA 的表达;流式细胞术测定 P-糖蛋白(P-gp)水平和caspase-3活性;细胞形态学和 FITC 标记的膜联蛋白 V/碘化丙锭(Annexin V-FITC/PI)双染色法检测细胞的凋亡。结果筛选出的 mdr1 siRNA-1和 mdr1 siRNA-2显著抑制 K562/ADM 细胞 mdr1的表达,mdr1 mRNA 的表达分别降低91.2%和82.0%,P-gp 水平下降74.1%和84.4%;增强 caspase-3活性,活化 caspase-3增加约40%;K562/ADM 耐药细胞对阿霉素诱导凋亡的敏感性增强,Annexin V-FITC/PI染色检测细胞凋亡率提高约60%。结论 siRNA 通过沉默 mdr1/P-gp 表达而逆转 K562/ADM 多药耐药细胞的凋亡抑制现象。
Objective To investigate the silencing effects of small interfering RNA (siRNA) on mdr1 gene expression and the reversal of apoptosis inhibition in leukemia multidrug-resistant cell line K562 / ADM. Methods K562 / ADM cells were used as target cells. Two siRNAs targeting mdr1 mRNA (mdr1 siRNA-1 and mdr1 siRNA-2) were selected and transfected into K562 / ADM cells by lipofectamine. Real-time fluorescence quantitative PCR was used to detect the expression of mdr1 mRNA; the P-gp and caspase-3 activities were determined by flow cytometry; the morphological and FITC-labeled Annexin V / propidium iodide Cell apoptosis was detected by double staining with Annexin V-FITC / PI. Results The mdr1 siRNA-1 and mdr1 siRNA-2 significantly inhibited the expression of mdr1 in K562 / ADM cells. The expression of mdr1 mRNA decreased by 91.2% and 82.0%, while the P-gp levels decreased by 74.1% and 84.4% The activation and activation of caspase-3 increased by about 40%. The sensitivity of K562 / ADM-resistant cells to adriamycin-induced apoptosis increased. The apoptosis rate of K562 / ADM cells increased by about 60% with Annexin V-FITC / PI staining. Conclusion siRNA can reverse the apoptosis-inhibitory effect of multidrug-resistant K562 / ADM cells by silencing mdr1 / P-gp expression.