Establishment of double-transfected MDCK Ⅱ cells expressing human organic anion transporting polypeptide 1B1 and multidrug resistance-associated protein 2 and identification of its functions / 复旦学报（医学版）

ABSTRACT

Objective To establish double-transfected Madin-Darby canine kidney (MDCK) [Ⅱ cells expressing human organic anion transporting polypeptide 1B1 (hOATP1B1) and multidrug resistanceassociated protein 2 (hMRP2)and to testify their functions,moreover,to study the transcellur transport of indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyltryptophan (1-MT) in the transfectants.Methods hOATP1B1/hMRP2 eukaryotic vectors pVITRO2-SLCO1B1-ABCC2 was obtained by genetic engineering method and then transfected into MDCK cells.Stably expressed MDCK cells were screened by using the geneticin G418.Real-time PCR,Western blot analysis and immuno fluorescent confocal microscopy were used to verify the proteins expression.Transport of the representative substrate pravastatin in different pH values and substrate concentrations and 1-MT were evaluated using the double transfectants.Results MDCK-OATP1B1/MRP2 was successfully established.Pravastatin displayed the optimal transcellular transport when pH value was 6.5.Transport of pravastatin demonstrated the concentration-dependent in the concertation range of 0) to 500 μmol/L.Transport of 1-MT showed no significant difference in MDCK cells and transfectants.Conclusions MDCK-OATP1B1/MRP2 was successful established;1-MT was not the substrate of OATP1B1 or MRP2 protein;and the eatablished double transfectant cell lines can be used to evaluate OATP1B1/MRP2-medicated transport of xenobiotics (e.g.new drug candidates) and endogenous compounds (e.g.bilirubin).