基于RNA干扰技术的外排转运体体外模型及评价  

The evaluation of efflux transporter model based on RNA interference technology in vitro

作　　者：孔令雷 阳海鹰 原梅 庄笑梅 李桦 

机构地区：[1]北京毒物药物研究所,抗毒药和毒理学国家重点实验室,北京100850

出　　处：《药学学报》2015年第9期1122-1127,共6页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81302760);中国博士后基金资助项目(2013M542510);国家“重大新药创制”科技重大专项资助项目(2008ZXJ09006001,2012ZX09301003-001)

摘　　要：为建立多药耐药相关蛋白2(MRP2)和P-糖蛋白(P-gp)的体外RNA干扰模型,应用化学合成的si RNA转染Hep G2细胞,并用实时PCR和Western blot检测m RNA和蛋白的表达变化,应用MRP2和P-gp底物罗丹明和甲氨蝶呤进行功能评价确定模型是否成功。si RNA筛选结果显示,MRP2 si RNA-3或P-gp si RNA-2能够显著地抑制MRP2或P-gp的m RNA表达,抑制率分别为68%和84%;MRP2 si RNA-3或P-gp si RNA-2(80 nmol·L-1)作用48 h能够明显地抑制MRP2或P-gp的蛋白表达,抑制率分别为62%和70%,同时不影响其他转运蛋白的表达;给予MRP2或P-gp底物甲氨蝶呤或罗丹明孵育,发现MRP2 si RNA-3或P-gp si RNA-2(80 nmol·L-1)作用48 h能够显著增加甲氨蝶呤或罗丹明的细胞内浓度,表明其外排受到抑制。以上结果表明利用化学合成的si RNA能够显著地抑制细胞MRP2和P-gp的表达和功能,提示细胞干扰模型建立成功。In the present study, the specifically knockdown models of P-gp or MRP2 were constructed by using a series of chemically synthesized small interfering RNA（si RNA） in vitro. The expression of P-gp and MRP2 was measured by real-time PCR and Western blot, and the function was evaluated by applying P-gp and MRP2 substrate, rhodamine and methotrexate. The results showed that MRP2 si RNA-3 or P-gp si RNA-2 significantly decreased the m RNA expression of MRP2 or P-gp, the inhibition ratio was 68% or 84%; MRP2 si RNA-3 or P-gp si RNA-2 at a dose of 80 nmol·L-1 significantly reduced the protein expression of MRP2 or P-gp at 48 h after treatment, the inhibition ratio was 62% or 70%. Meanwhile, other transporters were not influenced by si RNA. When pretreatment with MRP2 si RNA-3 or P-gp si RNA-2, the efflux of methotrexate or rhodamine decreased significantly and the intra-cellular concentration increased. The results suggested that chemically synthesized si RNA could significantly inhibit the expression and function of MRP2 and P-gp, and the model of RNAi in vitro could be used to evaluate the role of efflux transporters in transportation of drugs.

关 键 词：RNA干扰 小干扰RNA P-糖蛋白 多药耐药相关蛋白2 

分 类 号：R963[医药卫生—微生物与生化药学]