[利用短发夹RNA表达载体逆转多药耐药人白血病细胞系K562/ADM中mdrl基因依赖性多药耐药]

[Reversal of mdrl gene-dependent multidrug resistance in multidrug resistance human leukemia cell line K562/ADM using short hairpin RNA expression vectors].

作者信息

Gan Hui-zhu, Zhang Gui-zhen, Lu Zhen-xia, Pu Li-sha, Yang Shao-juan, Gao Shen, Zheng De-ming

机构信息

Department of Hematology, China-Japan Union Hospital, Jilin University, Changchun 130031, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2007 Jun;28(6):383-7.

DOI:

PMID:17939403

Abstract

OBJECTIVE

To explore the role of reversal multidrug resistance (MDR) using short hairpin RNA (shRNA) expression vectors in multidrug resistance human leukemia cell line K562/ADM.

METHODS

The oligonucleotides with 19-mer hairpin structure were synthesized. The shRNA expression vectors were constructed and introduced into K562/ADM cells. Expression of mdr1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western blot. The apoptosis and sensitivity of the K562/ADM cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscope (LCSM).

RESULTS

In positive clones of K562/ADM cells stably transfected with pSilencer 3.1-HI neo mdr1-A and mdr1-B shRNA expression vectors, RT-PCR showed that mdr1 mRNA expression was significantly reduced to 35.9% (P < 0.05), 27.5% (P < 0.01), respectively. Western blot showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 79-fold to 38-fold (P < 0.05), 30-fold (P < 0.01) respectively. Furthermore, the fluorescence intensity of K562/ADM cells was increased significantly compared with the control. shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The percent of the apoptosis cell was significantly enhanced to 18.1% (P < 0.05) , 54.4% (P < 0.01) respectively.

CONCLUSIONS

shRNA expression vectors can effectively reverse MDR, and restore the sensitivity of drug-resistance K562/ADM cells to conventional chemotherapeutic agents.

摘要

目的

探讨利用短发夹RNA（shRNA）表达载体逆转多药耐药（MDR）在多药耐药人白血病细胞系K562/ADM中的作用。

方法

合成具有19个核苷酸发夹结构的寡核苷酸。构建shRNA表达载体并导入K562/ADM细胞。通过逆转录聚合酶链反应（RT-PCR）评估mdr1 mRNA的表达，通过蛋白质免疫印迹法测定P-糖蛋白（P-gp）的表达。分别通过流式细胞术和甲基噻唑基四氮唑（MTT）法对K562/ADM细胞的凋亡情况及对阿霉素的敏感性进行定量分析。通过激光共聚焦扫描显微镜（LCSM）检测细胞柔红霉素蓄积情况。

结果

在稳定转染pSilencer 3.1-HI neo mdr1-A和mdr1-B shRNA表达载体的K562/ADM细胞阳性克隆中，RT-PCR显示mdr1 mRNA表达分别显著降低至35.9%（P<0.05）、27.5%（P<0.01）。蛋白质免疫印迹法显示P-gp表达受到显著且特异性的抑制。对阿霉素的耐药性分别从79倍降至38倍（P<0.05）、30倍（P<0.01）。此外，与对照组相比，K562/ADM细胞的荧光强度显著增加。shRNA载体显著增强了细胞柔红霉素的蓄积。凋亡细胞百分比分别显著提高至18.1%（P<0.05）、54.4%（P<0.01）。