Establishment of stably-transfected cell line with α2B-adrenergic receptor and enhanced green fluorescent protein labeled nucleus factor of activated t cells 2 / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE: To establish a stably-transfected cell line with adr adrenergic receptorα2B-AR) and enhanced green fluorescent protein (EGFP) labeled nucleus factor of activated T cells 2 (NFAT2) (EGFP-NFAT2) in U20S. METHODS U20S cells that stably expressed EGFP-NFAT2(U20S-EGFP-NFAT2 cells) were transfected with pcDNA3.1 expressing hygromycin B (Hygro) resistance gene and α2B-AR gene (pcDNA3.1-Hygro-α2B-AR) recombinant plasmid. The transfected cells were selected by Hygro (200 mg · L-1) for 10 d before being screened on the nucleus translocation assay of EGFP-NFAT2. Then, the selected cells were evaluated by Z' factor for functional stability and azrAR expression in the selected cells was analyzed by quantitative real-time PCR (qRT-PCR) and Western blotting. Finally, the activity of α2B-AR on NFAT2 nucleus translocation was evaluated by α2-AR agonist dexme-detomidine hydrochloride (DMED) or antagonist atipamezole hydrochloride (ATI) treatment. RESULTS Recombinant plasmid pcDNA3.1-Hygro-α2B-AR was established and transfected to U20S cells stably expressing EGFP-NFAT2. In the stably-transfected No.12 cell strain, the relative nuclear translocation index (0.445) was the highest among the selected cell lines, and Z' factors on three independent experiments were 0.664, 0.533 and 0.634, respectively. The mRNA expression of α2B-AR of No. 12 cell strain was detected by qRT-PCR and was about 140 times that of U20S-EGFP-NFAT2 cells (P<0.01) in 20 generations. The protein band of α2B-AR was also detected in No. 12 cell strain whereas no band of α2B-AR was detected in U20S-EGFP-NFAT2 cell by Western blotting. DMED concentration-depend-ently increased the relative translocation nuclear index in U20S-EGFP-NFAT2-α2B-AR cell [EC50= (2.616±0.121) nmol·L-1]. ATI concentration-dependency decreased the relative nuclear translocation index in U20S-EGFP-NFAT2-α2B-AR cell [IC50 =(89.05±0.22) nmol-L-1]. CONCLUSION The stably-transfected U20S-EGFP-NFAT2-α2B-AR cell line is established and can be used for high throughout screening of biased chemicals and the study on the mechanism of α2B-AR.