The Research Of HIF-1α SiRNA Recombinant Plasmid Inhibiting Retinal VEGF Expression In Diabetic Rats

ObjectiveThe vascular endothelial growth factor (VEGF) is the main growth factor which induced neovascularization in retina. We use RNA interference technique to inhibit VEGF. To evaluate the inhibitory effect of small interfering RNA (siRNA) targeting hypoxia inducible factor-1α (HIF-1α) on the expressions of vascular endothelial growth factor (VEGF) in diabetic rats.MethodsThis study included2parts:1.Randomized controlled trial was performed. HIF-1α siRNA of rat was designed and cloned into the eukaryotic expression vector pSilencer2.1-U6neo to construct the recombinant plasmid. After transformed into E.coli cells, the recombinant plasmid were confirmed by restrict enzyme digestion and sequence analysis. Monkey microvascular endothelial cells were cultured in vitro and divided into gene therapy group and vector group. VEGF protein were measured by western blot.2. There were totally one hundred and twenty-four male SD rats in which twenty-nine rats were choosen as normal control group and ninty-seven rats were diabetic rats group which was induced by injection streptozotocin (STZ).Pre-transfected normal group and DR group were randomly selected three rats for the retina, the remaining diabetic rats were randomly divided into there groups including diabetic retinopathy group (DR group), empty vector group and gene therapy group (HIF-1α siRNA group). Empty vector and gene therapy group was intravitreal injection Liposome mediated empty vector plasmid and HIF-1α siRNA plasmid respectively. Normal control group and DR group were not transfected. HE staining and FITC-dextran fluorescent angiography were used to assess the retinal vascular pattern.The VEGF mRNA levels in retinas were measured by Real-time RT-PCR and VEGF protein were measured by immunohistochemical. Significant differences between groups were evaluated by One-way ANOVA and LSD-t test. ResultsHIF-la siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. Fluorescent angiography shown a large number of micro-aneurysms,String-of-beads, high-fluorescein leakage and large non-perfusion area in diabeitic retinopathy.Proliferative retinal vascular endothelial cell can be found from the HE staining.It also verified angiogenesis budsand and angiogenesis cluster vertically growing out of the internal limiting membrane. Real-time RT-PCR revealed that the expression of VEGF mRNA was faint in the normal group, but increased obviously in the DR group (P<0.05).There was no statistically significant between DR group and vector group (P>0.05). The expression of VEGF mRNA in HIF-la siRNA group were obviously decreased compared with DR group. VEGF mRNA level was reduced by32.8%、43.6%、47.7%、50.9%at24h、48h、2h、1w. Immunohistochemistry revealed the expression of VEGF protein was weakly positive in normal control group, while the DR group and empty vector group were significantly increased.Compared with DR group and the empty vector group,gene therapy group was significantly decreased, the difference was statistically significant (P<0.05).VEGF protein level was reduced by27.4%、40.6%、47.5%、64.5%at24h、48h、72h、1w.ConclusionHIF-1α siRNA recombinant plasmid can effectively inhibit the expression of VEGF protein of the monkey retinal microvascular endothelial cells. Successfully constructed the early retinal neovascular models in diabetic rats.Intravitreal injection HIF-la siRNA recombinant plasmid can effectively inhibit the expression of VEGF in diabetic rats. New gene therapy may provided to control new vessels of diabetic retinopathy.