Stable expression and function of rat mu-opioid receptor in PC12 cells by lentivirus vector / 国际药学研究杂志

ABSTRACT

Objective To stably express rat mu-opioid receptor (rMOR) in PC12 cells with characteristics of neurons. Methods After the lentiviral vector pBPLV-rMOR-eGFP was constructed, the lentivirus was packaged and used to infect the PC12 cells. PC12 cells stably expressing rMOR was screened by the flow cytometry and the limiting dilution assay. The affinity and quantity of rMOR protein expressed in the PC12 were verified by radio-ligand binding assay. The function of rMOR was analyzed by cAMP overshooting. Results The eGFP protein in PC12-rMOR cells infected by the lentivirus could be clearly shown by the fluorescence microscope. Cell lines grew normally after every clone was enlargedly cultured. The affinity (Kd) and quantity (Bmax) values of rMOR were (0.51 ± 0.07) nmol/L and (1.58 ± 0.15)pmol/mg protein respectively in 3H-diprenorphine binding assay. The cAMP content increased (255 ±25.2) % after naloxone precipitated in chronic morphine-treated cells. Exogenous agmatine could dose-dependently inhibit the overshooting of cAMP by naloxone precipitated. Conclusion We have successfully established the PC12 cell model co-expressing stably rMOR and I1 style imidazoline receptor(I1R) without α2 adrenergic receptor, expressing properties of neurons, which is a good cell model in vitro for investigating the neural molecular mechanism of opioid addition and regulating the opioid receptor function by the system of agmatine and I 1 Rin the future.