Construction of VEGF small hairpin RNA expression plasmid and inhibition of VEGF expression in rat vascular endothelial cells
AIM: To construct short hairpin RNA(shRNA) eukaryotic expression plasmid of rat vascular endothelial growth factor( VEGF) and to explore its effects on VEGF expression in rat vascular endothelial cells (ECs). METHODS:Three different shRNA oligonucleotides targeting the coding sequence of rat VEGF mRNA were designed and cloned into pSilencer2. 0 U6 plasmid,respectively. The recombinant plasmids were then transfected into ECs via Lipofectamine 2000. Expression of VEGF was detected by RT-PCR and Western blot. RESULTS: VEGF shRNA expression plasmids were successfully constructed and identified by restriction enzyme and sequence analysis. VEGF expression was down-regulated by three VEGF shRNA plasmids and VEGF shRNA3 plasmid showed stronger inhibitory effect. The expression of VEGF mRNA was silenced by 71.91% and the expression of VEGF protein was silenced by 67. 86% at 72 h. CONCLUSION: The VEGF shRNA expression plasmids are successfully constructed and effectively inhibit the expression of VEGF mRNA and protein in ECs. The results of our study provide a basis for the inhibition of corneal neovascularization by RNA interference.
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万方数据
维普
