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To establish oxygen-induced retinal neovascularization in mice and to detect the expression of mTERT in mice.It was an experimental study Establishment of oxygen-induced retinal neovascularization in mice. Thirty-two 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group without restriction of gender. In oxygen-induced retinopathy group, 16 mice were exposed to 75% +/- 2% oxygen for 5 days and then to room air; In control group, 16 mice were raised in room air. Observation of the retinal neovascularization. On the postnatal day 19, The mice's vena caudalis were perfused with 2% Evens blue solution. Eyeballs were enucleated and fixed in 4% paraformaldehyde for half an hour. Then the retina was separated and flat-mounted on the slide. The morphologic changes of retinal vessel were observed and captured under fluorescence microscope. Histological observation and vascular endothelial cells counting. The eyeballs were enucleated and then fixed. After paraffin imbedding, 4 microm serial slices, hematoxylin-eosin staining, select one section every 60 microm to count the endothelial cell nucleus that break through the inner limiting membrane. Expression of mTERT mRNA were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). In the each group, the retina were all carefully dissected on the postnatal day 19. The total RNA was isolated and cDNA was synthesized before RT-PCR was performed. The PCR products were separated by 2% agarose gel electrophoresis and photographed. Expression of mTERT mRNA were confirmed by Real-time PCR The total RNA was isolated and cDNA was synthesized (The same procedure as RT-PCR). Fluorescent real-time quantitative polymerase chain reaction system (total 20 microl) was made. The Fluorescent signals were detected at 60 degrees C. The expression of mTERT were confirmed by immunohistochemistry. At P19, 4 microm cross sections were made in the hyperoxia-exposed and normal retinas. Sections were incubated with rabbit anti-Human/Mouse/Rat Telomerase 60 minutes at 37 degrees C. Anti-rabbit immunoglobulin G, depending on the primary antibody, was used as a secondary antibody for 30 min. Peroxidase activity was detected with the substrate diaminobenzidine. Permanent slides were covered with a 1.5 mm thick cover slip, examined using a light microscope and photographed.The central retina was nonperfused region at P12. The most of the central retina showed almost no perfusion and the radial vessels appeared tortuous and dilated at P14. Retinal neovascularization occurred at maximum between postnatal day 17 and postnatal day 19. Paraffin tissue slice with hematoxylin-eosin staining showed that in the control group the average counts of vascular endothelial cells which break through the inner limiting membrane were hardly seen, but in hyperoxia group were noticeably more than in the control group. Reverse-transcription polymerase chain reaction (RT-PCR) results: the mRNA of mTERT and bFGF in the retinopathy group were higher than in the control group (P0.05). Real-time PCR results: the expression of mTERT mRNA in the retinopathy group was noticeably higher than in the control group (F = 173.104, P0.05). Immunohistochemical staining showed that mTERT protein were positive in the retinal neovascularization of the hyperoxia group, but were negative in the retinal vessel of the control group.Telomerase reverse transcription and angiogenic correlation factors were up-regulated in a mouse model of oxygen-induced retinopathy, which may have therapeutic potential in the treatment with the neovascularization in retinopathy. |
