| Objective:The retinal neovascularization diseases, which result from pathological angiogenesis, are among the most common causes of blindness. The process of angiogenesis involves cellular and morphological changes, including endothelial cell proliferation, migration, and vascular tube formation. The p21WAF1/CIP1(p21) protein negatively regulates the cell cycle by binding to and inhibiting the activity of cyclin-dependent kinases (CDKs)-a family of proteins required for the transition from the G1-(interphase) to the S-(DNA synthesis) phases of the cell cycle. Inhibition of CDK2and cyclinE results in cell cycle arrest at the G1-S phase interface. In this study, firstly we established the mice model of oxygen-induced retinal neovascularization, and detected the expression of p21, CDK2and cylin E mRNA and protein, and discuss the role of p21in the pathogenesis of retinal neovascularization; secondly we transfected adenoviral vector-mediated delivery of p21to RF/6A and retina of the mice with retinal neovascularization, and discuss the inhibitory effect of adenoviral vector-mediated delivery of p21on RF/6A cell proliferation, migration and retinal neovascularization.Methods:(1) Ninety-six C57BL/6J mice at the age of7days were divided into two groups (experimental and control group) randomly. The model of oxygen-induced retinal neovascularization was establishe according to Smith protocol in experimental group. Retinal neovascularization was investigated on flat-mouts after fluorescence angiography and histopathology at the age of12,17and22days respectively. The expression of p21, CDK2and cyclin E mRNA and protein in retina were measured by RT-PCR and Western blot at the same time.(2) RF/6A cells were cultured in vitro, and were divided into phosphate buffered solution (PBS) group, adenoviral vector-mediated delivery of p21(Ad-p21) group and adenoviral vector-mediated delivery of non-target control (Ad-NC) group. The cell cycle distribution was analyzed by flow cytometry. Matrigel was used in endothelial-cell tube formation. The migration were detected by transwell. The expression of p21, CDK2and cyclin E mRNA and protein in RF/6A cells were measured by RT-PCR and Western blot respectively.(3) Eighty C57BL/6J mice at the age of7days were divided into control, phosphate buffered solution (PBS), Ad-p21and Ad-negative control (Ad-NC) groups randomly. The oxygen-induced retinal neovasculaiton model was induced by Smith protocol in PBS, Ad-p21and Ad-NC groups. At the age of11days, the mice among PBS, Ad-p21and Ad-NC groups received intravitreal injection of1ul PBS, Ad-p21and Ad-NC respectively. At the age of17and22days, retinal neovascularization was investigated on flat-mouts after fluorescence angiography and histopathology. At the age of17days, the expression of p21, CDK2and cyclin E mRNA and protein in retina were measured by RT-PCR and Western blot respectively.Results:(1) There were large areas of non-perfusion and fluorescein leakage in the retina of experimental group. The expression of p21mRNA and protein in experimental group were lower than that in control group, the difference between the two groups was significant. But the expression of CDK2mRNA and protein in experimental group were higher than that in control group, the difference between the two groups was significant.(2) The cell cycle distribution showed that the proportion of G0/G1cells in Ad-p21group was apprently higher than that in PBS and negative control group, the difference among these three groups was significant. Transwell results showed that the number of cells which passed the transwell in Ad-p21group was apprently less than that in PBS and negative control group. The number of endothelial-cell tubes in Ad-p21group was apprently less than that in PBS and negative control group. The expression of p21mRNA and protein in Ad-p21group were higher than that in PBS and negative control group. But the expression of CDK2and cyclin E mRNA and protein in Ad-p21group were higher than that in PBS and negative control group.(3) Compared with PBS and Ad-NC groups, the retinal non-perfusion areas, neovasculartion and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly. The expression of p21mRNA and protein in Ad-p21group were higher than that among the other three groups; the expression of CDK2and cyclin E mRNA and protein in Ad-p21group were lower than that among the other three groups.Conclusions:(1) p21may play an important role in the development of retinal neovascularization. The decline of p21expression induced endothelium proliferation may be the main reason.(2) The p21mRNA and protein can stably express in RF/6A cells after Ad-p21transfection. Ad-p21can inhibit the proliferation and migration of RF/6A cells by increasing the expression of p21.(3) Ad-p21can inhabit the proliferation of retinal vascular endothelial cells, and then inhabit retinal neovasculaiton by upregulating the expression of p21and downregulating the expression of CDK2and cyclin E. |
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