Generation Of Novel ABC Transporters Model And Its Application To Metabolic Study

The ATP-binding cassette(ABC)transporter family is one of the largest membrane transporter families,affecting the absorption,distribution,metabolism and excretion of drugs.The most studied subtypes of ABC transporter family are Pglycoprotein(P-gp),breast cancer resistance protein(BCRP)and multidrug resistanceassociated proteins(MRPs).Since current in vitro and in vivo models still have many defects,there is an urgent need to establish new models and methods.In this study,we presented a novel BCRP/MRP2 model based on intestinal 3D organoids.The crypts were isolated from small intestine of the mouse,and cultured to develop intestinal 3D organoids.Subsequently,we detected the expressions of BCRP and MRP2 in 3D organoids at mRNA and protein levels.Finally,the function of BCRP/MRP2 in 3D organoids was validated by fluorogenic substrates and positive inhibitors of BCRP/MRP2.A rapid and efficient model has been successfully established to study the function of BCRP and MRP2 in vitro and screen inhibitors of BCRP and MRP2.In terms of model construction in vivo,this thesis validated the Pgp(Mdr1a/1b)knockout rat model and carried out related metabolic studies.Western blot results showed that P-gp was completely absent in liver,small intestine,brain and kidney of P-gp knockout rats.Further pharmacokinetic studies of digoxin,a typical substrate of P-gp,confirmed the deficiency of P-gp in vivo.At the mRNA level,we investigated whether compensatory changes occur in the cytochrome P450 enzyme 3A subfamily and drug transporter-related genes in the liver,intestine,kidney,and brain of P-gp knockout rats compared to wild-type rats.P-gp knockout rats can be used to study P-gp function,screen P-gp substrates and inhibitors.In summary,this thesis innovatively constructed a BCRP/MRP2 model based on intestinal 3D organoids in vitro and a P-gp knockout rat model in vivo,which provided a powerful tool for studies of ABC transporters.