基于RNA干扰技术的外排转运体体外模型及评价

发布时间：2019-06-24 16:28

【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。
[Abstract]:In order to establish an in vitro RNA interference model of multidrug resistance-related protein 2 (MRP2) and P-glycoprotein (P-gp), Hep G2 cells were transfected with chemically synthesized si RNA, and the expression of m-RNA and protein was detected by real-time PCR and Western blot. The functional evaluation of rhodamine and methotrexate using MRP2 and P-gp was used to determine whether the model was successful. The results showed that the expression of MRP2 or P-gp was inhibited by MRP2 si RNA-3 or P-gp si RNA-2, and the inhibition rate was 68% and 84%, respectively. The expression of MRP2 or P-gp was inhibited by MRP2 si RNA-3 or P-gp si RNA-2 (80 nmol 路 L-1). The inhibition rate was 62% and 70%, respectively. At the same time, the expression of other transporters was not affected; either the MRP2 or P-gp substrate methotrexate or rhodamine was given, and it was found that MRP2 si RNA-3 or P-gp si RNA-2 (80 nmol 路 L-1) could significantly increase the intracellular concentration of methotrexate or rhodamine, indicating that its efflux was inhibited. The results showed that the expression and function of the cell MRP2 and P-gp were significantly inhibited by the chemical synthesis of si RNA, suggesting that the cell interference model was successfully established.
【作者单位】：北京毒物药物研究所抗毒药和毒理学国家重点实验室;
【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)
【分类号】：R963

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