Construction of shRNA expressing plasmid targeted rat NogoB and observation of the effection about NogoB on hepatic stellate cells

OBJECTIVE: To construct shRNA expressing plasmid inhibiting rat NogoB and to observe its possible effect on rat primary hepatic stellate cells (HSCs) contraction. METHODS: Three pairs of shRNAs targeting different sequence of rat NogoB were designed and constructed into pSuper plasmid by DNA recombination technique. Culture-activated HSCs were transfected with NogoB-shRNA plasmids to scan the effective plasmid which could inhibit NogoB gene expression by Real-time PCR. And this depressant effect was also confirmed with Western blot. After NogoB was knocked-down effectively, ETA and ETB mRNA expression were assessed by Real-time PCR. RESULTS: Among the three pairs of recombinant plasmids, NogoB-shRNA2 plasmid could inhibit NogoB expression specifically. In HSCs, NogoB knockdown decreased the ratio of ETA and ETB. CONCLUSION: We constructed specific NogoB-shRNA expression plasmid successfully which might be involved in contraction of HSCs.