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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。
To establish an in vitro RNA interference model of multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp), chemically synthesized si RNA was transfected into Hep G2 cells and real-time PCR and Western blot were used to detect m RNA and Protein expression changes, the application of MRP2 and P-gp substrate rhodamine and methotrexate functional evaluation to determine the success of the model. The results of si RNA screening showed that MRP2 si RNA-3 or P-gp si RNA-2 could significantly inhibit the expression of MRP2 or P-gp m RNA with the inhibition rates of 68% and 84%, respectively; MRP2 si RNA-3 or P Gp si RNA-2 (80 nmol·L-1) for 48 h significantly inhibited MRP2 or P-gp protein expression, the inhibition rates were 62% and 70%, respectively, while not affecting the expression of other transporters; given MRP2 or P-gp substrate methotrexate or rhodamine incubation and found that MRP2 si RNA-3 or P-gp si RNA-2 (80 nmol·L-1) for 48 h can significantly increase methotrexate or rhodan Bright intracellular concentration, indicating that its efflux was inhibited. The above results show that the use of chemically synthesized si RNA can significantly inhibit the expression of MRP2 and P-gp cells and function, suggesting that cell interference model was established.