[Expression of mouse telomerase reverse transcription in a mouse model of oxygen-induced retinopathy].

OBJECTIVE: To establish oxygen-induced retinal neovascularization in mice and to detect the expression of mTERT in mice. METHODS: 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. RESULTS: 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 (P < 0.05). Real-time PCR results: the expression of mTERT mRNA in the retinopathy group was noticeably higher than in the control group (F = 173.104, P < 0.05). 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. CONCLUSIONS: 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.

[TERT-siRNA inhibits oxygen-induced retinal neovascularization in mice].

OBJECTIVE: To investigate the inhibitory effect of small interfering RNA (siRNA) targeting TERT on murine retinal neovascularization and explore the feasibility of potential therapeutic approach in retinal vascular disease. METHODS: Two recombinant plasmids TERT siRNA (pSIREN-mTERT-1) and negative plasmid (pSIREN-mTERT-N) were constructed and 80 seven-day-old C57BL/6J mice were divided randomly into therapeutic group (A), negative plasmid group (B), oxygen-induced retinopathy group (C) and normal control group (D), 20 mice in each group. Group A, B and C were exposed to 75% +/- 2% oxygen for 5 days and then to room air, which induced mice retinal neovascularization. Groups A and B were injected two kinds of the above recombinant plasmid into the murine vitreous on the 12th day. The mice of group D were raised in normal oxygen circumstance. On the 19th day, 2% Evens blue angiography was used to observe the pattern of the retinal vascular. Expression of TERT mRNA were confirmed by reverse-transcription polymerase chain reaction (RT-PCR) and Real-time PCR. Histological observation and vascular endothelial cells counting were used to examine the effects of siRNA on the retinal neovascularization. RESULTS: Retinal flat after Evans blue angiography indicated that the vessels of group A formed a fine radial branching pattern, which was similar to normal mice. In group A, the retinal neovascularization reduced and the structure of retina were more regular than group B and C. At the same time the large vessels were distorted, neovascular clusters proliferated and fluorescence leaked in the middle and periphery area in group B and C. RT-PCR and Real-time PCR showed the expression of TERT mRNA was downregulated in group A compared with groups B and C (P < 0.05). Paraffin tissue slice with hematoxylin-eosin staining showed that the average counts of vascular endothelial cells which break through the inner limiting membrane in group A were less than groups B and C, the differences were significant (P < 0.05). CONCLUSION: Pathologic retinal neovascularization can be inhibited by specific TERT siRNA in vivo, which may be a novel efficient strategy against proliferative vasculopathies.