关键词: 维甲酸核受体γ，腺病毒载体，小干扰RNA，肝细胞分化

Abstract: Objective To construct and screen out the recombinant adenovirus vector expressing specific siRNA for mouse retinoic acid receptor-γ (RARγ) gene, and to detect the effect of inhibition of RARγ on hepatic differentiation of hepatic progenitor cells from post coitus day 14.5 mouse liver (HP14.5d). Methods Three pairs of siRNA sequence for mouse RARγ gene were designed and annealed in vitro to make double-stranded DNA, then cloned in SfiⅠ digested pSES-HUS vector and recombinated with the backbone vector pAdEasy-1 in E.coli BJ5183 to construct pAd-siRARγ plasmid. Ad-siRARγ was packaged in HEK293 cell line and used to infected HP14.5 cells. Real-time PCR and Western blot were performed to detect the expression of RARγ. 1 μmol/L ATRA was added to induce HP14.5d cells, ALB-driven Gaussian Luciferase (ALB-Gluc) activity assay and PAS staining were carried out to check hepatic differentiation. Results SiRNA fragments were confirmed to be correctly cloned in adenovirus vector by using PCR, endonuclease cutting and gene sequencing. Cloudiness amplification of RFP-positive cells was observed in HEK293 cell line at day 10 of adenovirus package. At 48 h after infection, more than 60% of HP14.5 cells were infected. Among three siRNA, Ad-siRARγ2, 3 effectively inhibited the expression of RARγ. ATRA induction could increase ALB expression and glycogen storage function of HP14.5d cells, However, Ad-siRARγ2,3 infection significantly reversed ATRA induced hepatic differentiation, ALB-Gluc activity and the ratio of PAS stained cells were statistically lower than that in induction group（p<0.05）. Conclusions Successfully constructed and screened out the recombinant adenovirus vector containing siRNA for mouse RARγ gene, which could effectively inhibit the expression of RARβ in HP14.5d cells and then reverse its hepatic differentiation with ATRA treatment.

Key words: retinoic acid receptor-γ, adenovirus vector, small interfering RNA, hepatic differentiation