关键词: 细胞缝隙连接, CX26/CX32, Tet-On调控系统, HeLa细胞, 诱导表达

Abstract: Objective To establish a specific cell model for fast and efficiently measuring the effect of candidate drug on gap junction (GJ), which will provide an ideal experimental tool for both the drug discovery targeting GJ and the specific research for GJ. Methods The bidirectional vector pBI plasmid under the control of a bidirectional doxycycline-inducible promoter was constructed. The Tet-on HeLa cells were transfected with CX26/CX32 cDNA and the cell model stably expressing CX26/CX32 and functional heteromeric GJ channels were established. Cells were cultured in the presence and absence of doxycycline (Dox, 1 μg/mL), the expression of CX26 mRNA and protein in Tet-on HeLa cell were assayed by RT-PCR and Western blot, respectively. The GJ function between adjacent HeLa cells was detected by dye transfer assay, and the proliferation of HeLa cells was measured using sulforhodamine B (SRB) assay. Upon the above cell model, the effects of GJ inhibitor, 2-aminoethoxydiphenyl borate (2-APB), and activator, retinoid acid (RA), on the function of GJ were also observed. Results HeLa cells expressed no endogenous CX, and expression of CX26 mRNA and protein in Tet-on HeLa cells could be induced by the addition of Dox in the culture medium. Formation of functional GJ due to CX induction by Dox was observed as evidenced by the dye transfer assay. 2-APB (50 μmol/L) decreased dye spread through GJs composed of CX26/CX32 in HeLa cells with a GJ inhibition rate of 51.3% (P＜0.01); while RA (10 μmol/L) increased dye spread through GJs between adjacent HeLa cells with a GJ enhancement rate of 60.3% (P＜0.01). Conclusion Tet-on HeLa cell model stably expressing CX26/CX32 induced by Dox was successfully established, providing an useful technology for both the drug discovery targeting GJ and the specific research of GJ modulation by candidate drugs.

Key words: gap junction, CX26/CX32, Tet-On regulatory system, HeLa cell, inducible expression