pEGFP-NTCP稳定转染HEK293细胞系的建立及鉴定

摘要

目的：高效快速地建立稳定高表达钠离子－牛磺胆酸共转运多肽（sodium taurocholate cotransporting polypeptide， NTCP）的HEK293细胞系。方法构建可表达EGFP-NTCP融合蛋白的pEGFP-NTCP重组质粒，并利用FuGENE 6转染试剂将重组质粒转染至HEK293细胞中。用荧光显微镜观察绿色荧光蛋白表达情况，挑选转染细胞进行14 d G418筛选和单克隆培养，以获得稳定转染细胞株。用RT-PCR法、qRT-PCR法和Western blot技术检测稳定转染细胞及正常细胞中NTCP mRNA和蛋白的表达情况，并进一步用超高效液相色谱－串联质谱法（UPLC-MS／MS）考察稳定转染细胞的牛磺胆酸摄取能力。结果使用本文优化的方法，细胞转染率可达90％。RT-PCR、qRT-PCR、Western blot和牛磺酸摄取实验结果显示：相对于正常组，在荧光显微镜下呈绿色荧光的转染细胞；其NTCP表达均显阳性（P＜0.01）；且稳定转染细胞的牛磺胆酸摄取能力明显升高（P＜0.05）。结论高效快速地建立了稳定高表达NTCP的HEK293细胞系，为胆酸衍生物摄取机制研究奠定了基础。%Abstrate:Aim 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.