Establishment and identification of HEK293 cell lines with stable and high expression of NTCP / 中国药理学通报

ABSTRACT

AbstrateAim To construct HEK293 cell line with stable and high expression of sodium taurocholate cotransporting polypeptide (NTCP ) efficiently and rapidly.Method Vector expressing EGFP-NTCP fusion protein was constructed and verified by DNA sequencing.The pEGFP-NTCP expression vector was transfected into HEK293 cells by FuGENE 6 transfection reagent. The transfected cells with high expression of green fluorescent protein were selected using fluorescence microscope for screening of G418 for 14 days to obtain cell lines stably and highly expressing NTCP.NTCP expression was detected by RT-PCR,qRT-PCR, Western-blot and the uptake experiment of taurocholic acid.Re-sult RT-PCR,qRT-PCR,Western-blot and the uptake experi-ment revealed that compared to the control cells,the expression of NTCP was significantly positive (P<0.01)in stable trans-fected cells showing green fluorescence (P<0.05 ).Conclusion The HEK293 cell line with stable and high expression of NTCP has been established efficiently and rapidly,which provides a cellular model for the study of the mechanism of the uptake of bile acid derivatives.