Abstract: Objective To construct recombinant lentiviral vectors to deliver short hairpin RNA of rat Adrb3 (rAdrb3) gene and observe the effect of rAdrb3 gene expression on rat vascular smooth muscle cells (VSMC). Methods Two rAdrb3 gene shRNA oligonucleotide sequences were designed, and lentiviral interference plasmids were constructed and sequenced. The lentivirus was packaged by co-transfecting 293T cells with three plasmids, and then the lentivirus titers were measured. Rat VSMC were randomly divided into 4 groups, Normal group, Lv-rAdrb3-shRNA-control group, Lv-rAdrb3-shRNA-1 group and Lv-rAdrb3-shRNA-2 group. The cells were collected at the fifth day after infection. Total RNA and protein were extracted. The expression of rAdrb3 mRNA on rat VSMC was detected by real-time PCR. The expression of rAdrb3 protein on rat VSMC was detected by Western blot assay. Results The recombinant lentivirus was successfully constructed and the titer after purification was 2 × 108 TU/mL. The infection efficiency of the recombinant lentivirus on rat VSMC was 80%. Compared with the Normal group, the silencing efficiencies of rAdrb3 mRNA were 87.18% and 65.27% for Lv-rAdrb3- shRNA-1 group and Lv-rAdrb3-shRNA-2 group (P < 0.05). Compared with the Normal group, the silencing efficiecies of rAdrb3 protein were 85.57% and 70.04% for Lv-rAdrb3-shRNA-1 group and Lv-rAdrb3-shRNA-2 group (P < 0.05). Conclusion The recombinant lentiviral vectors to deliver short hairpin RNA of rAdrb3 gene, which can significantly inhibit the expression of Adrb3 gene on rat VSMC, are constucted successfully.

Key words:  receptors, adrenergic, beta-3, RNA interference, lentivirus, chronic heart failure, vascular smooth muscle cells