shRNA表达载体对耐阿霉素的人红白血病细胞株P-gp表达的抑制作用

Inhibition of P-gp Expression in Amycin-resistant Human Erythroleukemia Cell Strain K562/ADM by shRNA Expression Vector

目的探讨靶向mdr1基因的shRNA表达载体对耐阿霉素的人红白血病细胞(K562/ADM)P-gp表达的抑制作用。方法合成靶向mdr1基因的短发夹shRNA表达载体,转染K562/ADM细胞。利用RT-PCR检测转染细胞中mdr1基因mRNA,Westernblot、免疫细胞化学和流式细胞术分别检测P-gp的表达。结果在瞬时转染pSilencerTM3·1-H1neomdr1-A和mdr1-BshRNA表达载体的K562/ADM细胞中,mdr1基因mRNA转录分别减少到39·1%(P<0·05)和30·8%(P<0·01)。Westernblot、免疫细胞化学和流式细胞术检测显示P-gp表达被明显而特异地抑制。结论靶向mdr1基因的shRNA表达载体能够特异而有效地抑制耐药的人红白血病细胞中的P-gp表达。

Objective To explore the inhibitory effect of shRNA expression vector for mdrl gene on the expression of P-gp in amycin-resistant human erythroleukemia cell strain K562/ADM. Methods Transfect K562/ADM cells with the synthetic shRNA expression vector for mdrl gene. Determine the mdrl mRNA in transfected K562/ADM cells by RT-PCR,and expressed P-gp by Western blot, immunocytochemical method and flow cytometry. Results In the K562/ADM cells transiently transfected with pSilencer^TM 3. 1-H1 neo mdr1-A and mdr1-B shRNA expression vectors,the transcription levels of mdrl gene mRNA decreased to 39. 1% （P 〈0. 05） and 30. 8% （P 〈0. 01 ） respectively. Western blot,immunocytochemical method and flow cytometry proved that the expression of P-gp was significantly and specifically inhibited. Conclusion The shRNA expression vector for mdrl gene specifically and effectively inhibited the expression of P-gp in human erythroleukemia cells.