| ObjectiveObserving diabetic rats retina blood capillary change in form and study its influence to vascular endothelial growth factor receptors (VEGFRs,I.e.Flt-l,Flk-l) level with recombinant adeno-associated virus conducted pigment epithelium-derived factor (rAAV-CMV-PEDF) intervention in diabetic retinopathy rats, furthermore to comprehend the PEDF action mechanism of the antiangiogenesis and its influence to diabetic retinopathy.MethodsWe selected the healthy and male Wistar 60 rats that the body weight between at 200 and 250 gram, randomly divided into common groups, contrast groups and experimental groups, among the common and contrast groups were 15 rats, the experimental groups were 30 rats, and uniformly divided 3 time phases to part 1 month, 3 months and 6 months. To induce the contrast and experimental groups rats to diabetic rats, we injected streptozotocin(STZ) to contrast and experimental groups rats according to 60mg per kilogram. We respectively injected rAAV-CMV-PEDF and rAAV-CMV-GFP to right eyes and left eyes into all the experimental groups that have became to diabetic rats model. We executed the respective groups rats according to 1 month, 3 months and 6 months. To manifest the transfection capability of the rAAV, the retina stretched preparation technique was used to observe the expresstion of green fluorescent protein (GFP) in experimental group rats retina; The immunohistochemistry technique was used to detected Flt-1 and Flk-1 protein level change in diabetic rats retina with streptozotocin induced; The retina vessel stretched preparation was used to observe the diabetic rats retina capillary change in form, and to count endothelial cells (E), perithelial cells (P) and change in the two ratios.ResultsGFP was observed to express in experiment rats retinas and distribute at the whole layer after 1 month, significantly increased on the 3rd and 6th month. It manifested that rAAV possess more powerful transfection capability, rAAV could transfect PEDF to experiment rats eyes successfully, and PEDF could express persistently in eyes. The experimental group and control group expression of Flt-1 were both increased on the 3rd month and 6th month, but the control group were statistically significance (P<0.05), the experimental group were not statistically significance (P>0.05). The experimental group expression of the Flk-1 decreased on the 3rd month, significantly decreased on the 6th month, they were both statistically significance (P<0.05). The expression of control group of Flk-1 increased on the 3rd month, significantly increased on the 6th month, they were both statistically significance (P<0.05). The retina vessel stretched preparation manifested with the extension of the course of disease, the control group rats retinal capillary significantly change in form, but the experimental group were no significance change in form. The perithelial cells (P) population and E/P ratio of the common group and experimental group were no statistically significance (P>0.05), but the contrast group compared to common group, the perithelial cells(P) population decreased and E/P ratio increased, the difference was statistically significance on the 3rd month and 6th month (P<0.05). The contrast group compared to experimental group, the perithelial cells (P) population decreased and E/P ratio increased on the 6th month, the difference was statistically significance(P<0.05).ConclusionThrough rAAV-CMV-PEDF intervention, the diabetic rat model with the extension of the course of disease, the expression of Flk-1 in retina decreased, but the expression of Flt-1 was no statistically significance, the reduction of the perithelial cells (P) population was no statistically significance. We presumed that maybe the binding of PEDF to Flk-1, it suppressed the binding of VEGF to vascular endothelial cell, accordingly interrupted that the effect of VEGF contributing to vascular endothelial cell proliferation, therefore it maybe exist the tendency that the extrinsic source PEDF delay the progression. |
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