Study On Uptake Of OATP1B1 Genetic Polymorphism Effect On The Metabolism Of Tamoxifen And Its Metabolites

Objective: To establish overexpression lentivirus platform of OATP1B1(organic anion transporting polypeptides 1B1)wild type and mutant type genetic polymorphism in vitro,and using this platform to research and compare the uptake of tamoxien and its metabolites by mutating the 388 th and the 521 th bases.Methods: Extracted total RNA from human liver and reversed into cDNA,using PCR technology to get the OATP1B1 gene fragments.At the same time,by using enzyme digestion to cut the carrier GV358,ensured that the end sequence of amplification products were same with the sequence of the linearization cloning vector.To make a restructuring reaction system,compound linearization carrier with target gene amplification,realize the cyclization between the linearization and the target gene.Make the recombinant products amplification into DH5 alpha cells.To verify the sequence after extraction and purification,the mutation of OATP1B1388G(OATP1B1*1b)and521C(OATP1B1*1b)were obtained from the right ones.The recombinant plasmid and packaging plasmid pHelper1.0 and pHelper 2.0 were transfected into HEK293 T cells,packaging and producing the lentivirus,then harvest lentivirus for enrichment and purification.Maked the lentivirus conform to the requirements of the experiment,then infected the targeted HEK293 T cells.The expression of OATP1B1 messenger ribonueleie acid(mRNA)and Protein were detected by using Real-time quantitative PCR and western blot.To establish a methodology about HPLC-MS/MS for Z-tamoxien and its active metabolites(endoxifen).The study was divided into six group,firstgroup was HEK293 T cells;the second group was HEK293 T +drug;the third group was negative control lentivirus-HEK293T(HEK293T cells transfected with vector plasmid)+drug;the fourth group was OATP1B1*1a-HEK293 T +drug;the fifth group was OATP1B1*1b-HEK293T+drug;the sixth group was OATP1B1*5-HEK293T+drug.Take different concentrations of tamoxifen and endoxifen into the six groups for 24 hours and 48 hours.Use repeated freezing and thawing method with sterile water to split cells.Make use of the established HPLC-MS/MS methods to compare the differences in uptake.Results: 1)Detect the content of tamoxifen and endoxifen after 24 hours,the content of second group is similarity to the third group,which no statistical significance(P >0.05).The content of tamoxifen and endoxifen in the fourth group and the fifth group and the sixth group are remarkably higher than the second group,which is statistically significant(P<0.01).Make a conclution that tamoxifen and endoxifen can be uptaked into cells by transporter OATP1B1.2)After 24 hours,the content of tamoxifen and endoxifen in the fifth group compared with the fourth group,there is no statistical significance(P > 0.05).The content of tamoxifen and endoxifen in the sixth group is lower than the fourth group and thereis statistically significant(P<0.05).So it will inhibit the uptake ability of the transporter when the 521 th bases mutate from T to C.3)Detect the content of tamoxifen and endoxifen after 48 hours,the content of second group is similarity to the third group,which no statistical significance(P > 0.05).The content of tamoxifen and endoxifen in the fourth group and the fifth group and the sixth group are remarkably higher than the second group,which is statistically significant(P<0.01).4)After 48 hours,the content of tamoxifen and endoxifen in the fifth group compared with the fourth group,but there is no statistical significance(P > 0.05).The content of tamoxifen and endoxifen in the sixth group is lower than the fourth group and there isstatistically significant(P<0.05).The conent of drugs in every group were more and more with the increase of drug concentration and the extended of time,but the content of drug in cell lysis solution for 48 hours are higher than the content of drug in cell lysis solution for 24 hours,but there is no statistical significance(P > 0.05).Make a conclusion that OATP1B1 uptake tamoxifen and endoxifen will be saturated during 24 hours.Conclusion:The overexpression lentivirus cell platforms have been successfully constructed,including OATP1B1*1a-HEK29 T and OATP1B1*1b-HEK293 T and OATP1B1*5-HEK293 T cell model,the infection efficiency is 80% or more.The gene expression are high at mRNA and protein level.The tamoxifen and endoxifen can be uptaked into cells through organic anion transporter polypeptide1B1,and OATP1B1521T>C will inhibt the function of the transport protein,result in the content of drug in cell lysis liquid in OATP1B1*5+drug group is lower than in wild group+drug,and there is statistical significance(P<0.05).OATP1B1388A>G will make the content of drug in cell lysis liquid in OATP1B1*1b+drug and the wild group+drug similarity,and there is no statistical significance(P>0.05).