The Role And Mechanism Of15-lipoxygenase-1Gene Transfer On Oxygen Induced Retinal Neovascularization In Mice

Part One Construction, packaging, identification and expression of recombinant adenovirus carrying mouse15-Iipoxygenase-1genePurpose:To construct a recombinant adenovirus vector expressing mouse15-lipoxygenase-1(15-LOX-1) gene carrying enhanced green fluorescent protein (EGFP) tag.Methods:The mouse15-LOX-1gene was obtained by Polymerase chain reaction (PCR) amplification from cDNA library containing mouse15-LOX-1gene, and then cloned into eukaryotic expression vector pDC315-EGFP to construct the shuttle plasmid pDC315-15-LOX-1-EGFP. The shuttle plasmid pDC315-15-LOX-1-EGFP that carrying EGFP tag was cotransfected with the adenovirus skeleton plasmid pBHGlox-A El,3Ere into HEK293cell to obtain the recombinant adenovirus Ad-15-LOX-1-EGFP by site-specific recombination, which was detected by Western blot and EGFP expression respectively. The recombinant adenovirus was propagated by repeat infection of HEK293cell and purified by Adeno-XTM Virus Purification Kit, and then the virus titer was determined by the method of end-point dilution.Results:The recombinant adenovirus Ad-15-LOX-1-EGFP was constructed successfully, which was confirmed by Western blot and EGFP expression. After amplification and purification, the titer of recombinant adenovirus was1.25×1011PFU/ml.Conclusion:The recombinant adenovirus vector expressing mouse15-LOX-1gene carrying EGFP tag was successfully constructed by the method of homogenous recombination in cells. Part Two The expression and significance of15-lipoxygenase-1in a mouse model of oxygen induced retinopathyPurpose: To establish oxygen induced retinopathy (OIR) model in C57BL/6J mice by variable oxygen environment and explore the expression and significance of15-LOX-1on retinal neovascularization in oxygen induced retinopathy model.Methods:1867-day-old C57BL/6J mice were randomly divided into two groups that normal control group and OIR model group. The normal control group mice were raised in a normal oxygen environment while the OIR model group mice were place into an oxygen-regulated chamber under hyperopic condition (75±2)%O2for5days and were then kept in normoxic condition for a further5days to establish an animal model of oxygen induced retinopathy. OIR model group and normal control group mice were sacrificed at postnatal day17, both eyes were embedded in paraffin and performed hematoxylin and eosin (HE) staining, counting the endothelial cell nucleus which broke through the internal limiting membrane (ILM) in cross-sections. Both the OIR model and normal control group mice were performed on fluorescein retinal angiography for research of retina vascular morphological changes and retina neovascularization at postnatal day17. Both the OIR mode and normal control group mice were sacrificed at postnatal day7、9、12、14、17and21respectively, Real-Time PCR and Western Blot methods were used to detect the expression of15-LOX-1in normal control group retinal and on retinal neovascularization in OIR model group.Results:Compared with the normal control group there were large retinal neovascularization which structure and distribution was disordered and more endothelial cell nucleus which broke through the internal limiting membrane in the OIR model group (P<0.01). The expression of15-LOX-1in the OIR model group compared with in the normal control group were significantly decreased in gene and protein levels (p<0.05; p<0.05), and retinal15-LOX-1levels were negatively correlated with the progression of retinal neovascularization.Conclusion:The study indicated that the obvious decreased expression of15-LOX-1in a mouse model of oxygen induced retinopathy in gene and protein levels, which were high negatively correlated with the progression of retinal neovascularization.15-LOX-1may be a novel therapeutic target for ocular neovascularization diseases. Part Three The mechanism and inhibitory effects of15-lipoxygenase-l gene transfer on oxygen induced retinal neovascularization in miceObjective:To investigate the mechanism and inhibitory effects of15-lipoxygenase-1gene transfer on oxygen induced retinal neovascularization in mice.Methods:1087-day-old C57BL/6J mice were randomly divided into normal control group, induced model group, gene treated group and empty vector group.The mice with their mothers were rose in (75±2)%O2environment for5days and then returned to normoxia for5days to establish the oxygen induced retinopathy (OIR) model. At postnatal day12, the gene treated group was received intravitreous injection of Ad-15-LOX-1-EGFP at1.0μl, while the empty vector group was received the same volume of Ad-EGFP. The expression of green fluorescent protein was observed on flat-mounted retina by fluorescence microscopy2days after intravitreous injection. At postnatal day17, immunofluorescence staining method was used to detect the efficacy of15-LOX-1gene transfer on retinal tissue, Real-Time PCR and Western Blot methods were used to detect the mRNA and protein expression levels of15-LOX-1, peroxisome proliferator-activated receptor y (PPAR-y), vascular endothelial growth factor-A (VEGF-A) and vascular endothelial growth factor receptor2(VEGFR-2) in the retinal. The changes of retinal vessels, relative retinal non-perfusion and neovascularization areas were evaluated by FITC-dextran fluorescein angiography on flat-mounted retina. The number of endothelium cell nuclei breaking throuhgh the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section. Group differences were analyzed with one-way ANOVA followed by the Bonferroni post-test.Results:2days after intravitreous injection of Ad-15-LOX-1-EGFP, the expression of green fluorescent protein had been seen by fluorescence microscopy on flat-mounted retina. Immunofluorescence staining of retinal section revealed that15-LOX-1expression was primarily in the outer plexiform layer (OPL), inner nuclear layer (INL) and ganglion cell layer (GCL) retina. The15-LOX-0and PPAR-y protein and mRNA expression levels were higher in gene treated group than those in induced model group and empty vector group (P<0.01; P<0.01). On the contrary, VEGF-A and VEGFR-2expression levels were lower in gene treated group than those in induced model group and empty vector group (P<0.01; P<0.01). The relative retinal non-perfusion and neovascularization areas were significantly smaller, and the number of endothelium cell nuclei breaking through the ILM was obviously lower in gene treated group than those in induced model group and empty vector group (P<0.01; P<0.01).Conclusion:15-LOX-1gene transfer can decrease the oxygen induced retinal non-perfusion areas and inhibit the retinal neovascularization in mice via up-regulation of PPAR-γ and down-regulation of VEGF-A and VEGFR-2expression.