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

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

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-mdrl 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。

Objective To investigate the modulation for multidrug resistance cell line K562/A02 us- ing a specific siRNA against mdrl,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 pSilenee-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(IC_(50))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 pSilenee-mdr1,the expression of mdr1 mRNA in K562/A02 in was re- duced 71.5 % compared to the mock transfeetion,from(2.8±1.65)×10~8 copy/μg RNA to(3.9±2.37)×10~7 copy/μg RNA(P<0.01);While the expression of GSTπ mRNA in K562/A02 cell transfeeted with pSilenee-GSTπ was reduced 39.8% compared to the mock transfection,from(2.3±1.14)×10~5 copy/μg RNA to(5.4±2.45)×10~4 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.IC_(50)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 pSilenee-GSTπ can effectively reverse the multidrug resistance of cell line K562/A02.