多药耐药基因、谷胱甘肽S-转移酶特异性siRNA逆转K562／A02细胞多药耐药.

 目的　研究多药耐药基因（mdr1）、谷胱甘肽S-转移酶（GSTπ）特异性siRNA逆转K562／A02细胞多药耐药性。方法　以mdr1 mRNA第79～99和GSTπmRNA第308～327核苷酸为作用靶点，合成针对靶区域序列的siRNA，克隆入pSilence2.1-U6 neo，克隆产物为pSilence-mdr1和pSilence-GSTπ，脂质体介导下转染K562／A02细胞。用实时荧光定量PCR检测K562／A02细胞 mdr1和GSTπ mRNA的表达；流式细胞仪检测细胞凋亡；MTT法检测多柔比星的半数抑制浓度（IC50）。结果　经酶切、测序分析表明，成功构建siRNA真核表达载体。经pSilence-mdr1转染后的K562／A02细胞株mdr1 mRNA表达量下降了71.5 %，从（2.8±1.65）×108拷贝／μg RNA下降至（3.9±2.37）×107拷贝／μg RNA（P＜0.01）；同时pSilence-GSTπ 作用后，K562／A02细胞 GSTπmRNA表达量较显示空载体pSilence2.1-U6 转染组下降了39.8 %，从（2.3±1.14）×105拷贝／μg RNA下降至（5.4±2.45）×104拷贝／μg RNA（P＜0.01）；流式细胞仪显示空载体pSilence2.1-U6 转染组细胞凋亡率为（11.65±4.06）%，pSilence-mdr1和 pSilence-GSTπ共转染组细胞凋亡率为（44.98±11.27）%（P＜0.01）；空载体转染组耐药指数为23，pSilence-mdr1和pSilence-GSTπ共转染组耐药指数降低为7，IC50值从转染前的（1.16±0.38）mmol／ml下降为（0.33±0.04）mmol／ml（P＜0.01）。结论　siRNA 真核表达载体pSilence-mdr1、pSilence-GSTπ对K562／A02细胞株多药耐药性具有明显的下调作用。

A Study of the modulation for multidrug resistance cell line K562/A02 using a specific siRNA against mdr1, GSTπ

Objective　To investigate the modulation for multidrug resistance cell line K562/A02 us－ing a specific siRNA against mdr1, GSTπ. Methods　siRNA were synthesized targeting the coding region se－quences of mdrl (79～99 nt) and GSTπ(308～327nt) respectively，and cloned to plasmid pSilence2.1-U6. The cloned products pSilence-mdr1 and pSilence-GSTπ were transfected into K562/A02 cells. Expression of mdr1 and GSTπ mRNA were assayed by SYBR Green I real-time PCR. The apoptosis of cell line K562/A02 was examined by Flow cytometry , 50% inhibition concentration（IC50）of doxorubicin on K562/ A02 cell was deter－mined by MTT method. Results　The siRNA expression vector against mdr1, GSTπ mRNA was constructed successfully. After transfected with pSilence-mdr1, the expression of mdr1 mRNA in K562/A02 in was re－duced 71.5 % compared to the mock transfection, from (2.8±1.65)×108 copy/μg RNA to (3.9±2.37)×107 copy/μg RNA（P ＜0.01）; While the expression of GSTπ mRNA in K562/A02 cell transfected with pSilence-GSTπ was reduced 39.8% compared to the mock transfection, from (2.3±1.14)×105 copy/μg RNA to (5.4±2.45)×104 copy/μg RNA（P＜0.01）. The apoptosis rate of K562/A02 cell line transfected with pSilence2.1-U6 was(11.65±4.06)%, the apoptosis rate of K562/A02 cell line transfected with pSilence-mdr1 and GSTπ were(44.98±11.27)% (P <0.01). After transfected with pSilence-mdr1 and GSTπ, the resistance index (RI) of cell line K562/A02 trans－fected with pSilence2.1-U6 was 23, and RI of cell line K562/A02 transfected with pSilence-mdr1 and GSTπ was 7. IC50 of doxorubicin (ADR) on K562/A02 cell was decreased from (1.16±0.38) mmol/ml to (0.33±0.04) mmol/ml (P <0.01). Conclusion　pSilence-mdr1 and pSilence-GSTπ can effectively reverse the multidrug resistance of cell line K562/A02.