| Objectives:1.To construct hemeoxygenase-1(ho-1)over-expressed adeno-associated virus(AAV)vector,and then it is successfully transfected into rat retinal tissue.2.To study the protective effects of ho-1 over-expressed adeno-associated virus vector on retinal injury in rats.Methods:1.By constructing ho-1 over-expressed adeno-associated virus vector,and then determining the viral titer.2.By establishing a rat model of retinal damage,the tail vein injection of sodium iodate was used to induce the rat model of retinal damage.After the model was made,the eyeball of the rat was taken for pathological examination,to evaluate the situation of retinal structure damage,and to verify whether the model was successful.SD rats were divided into blank group,injury group,GFP group and HO-1 group.Aav-ho-1,AAV-GFP and normal saline were injected into the retina of HO-1 group,GFP group and injury group respectively,and the eyeballs of the rats were taken for examination after 3 weeks.3.HE staining was used to detect the pathological changes of retinal injury in rats,and the apoptosis of retinal tissue cells was detected by the gap terminal marker enzyme(TUNEL)mediated by deoxyribonucleotide terminal transferase(deoxyribonucleotide terminal transferase).The green fluorescence expression of retinal tissue was observed under fluorescence microscope.The expression levels of ho-1,caspase3 and bcl2 in four groups of rat retinal tissues were determined by Western Blot.Results:1.Successful construction of an adeno-associated virus vector with ho-1 over expression;2.Successful construction of the rat retinal degeneration model induced by sodium iodate;Green fluorescent protein was observed in retina under fluorescence microscope,indicating that ho-1 gene was successfully transfected into rat retinal tissue.3.HE staining:The injury group and the GFP group showed significant pathological damage to the rat retinal outer nuclear layer cells,which showed significant statistical difference compared with the blank group(P < 0.05).Compared with the blank group,the retinal structure of the ho-1 group was slightly changed,and the thickness of the outer nuclear layer was slightly thinner,with no significant statistical difference(P >,0.05).4.Wester-blot results showed that Compared with the blank group,ho-1 protein expression increased in the ho-1group,showing a significant statistical difference(P< 0.05).The expression of bcl-2 in ho-1 group was significantly increased,and the difference between the injury group and the GFP group was statistically significant(P < 0.05).Caspase-3 expression was reduced in the ho-1 group,and there was a significant statistical difference between the injury group and the GFP group(P < 0.05).5.TUNEL results showed that the retinal apoptotic cells in ho-1 group were reduced,and there was a significant statistical difference between the injured group and the GFP group(P < 0.05).Conclusion:1.AAV-HO-1 can be transfected into rat retinas by adeno-related virus packaging.2.Overexpression of HO-1 gene can inhibit the apoptosis of retinal tissue cells and has a protective effect. |
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