摘要
目的研究高氧诱导视网膜新生血管模型中小鼠端粒酶逆转录酶（TERT）基因表达水平是否有变化，为进一步研究视网膜新生血管疾病的预防和治疗提供新的靶点。方法实验研究。选取7d龄C57BL/6J新生小鼠32只，分高氧组和对照组，每组16只。高氧组小鼠以密闭氧箱内以75％±2％氧浓度饲养5d后置于正常氧浓度环境中，正常对照组小鼠于正常氧环境中饲养。于小鼠生后12、14及19d时分别取高氧组和对照组小鼠各2只（4只眼），经尾静脉行2％伊文思蓝溶液灌注并做视网膜铺片，荧光显微镜下观察视网膜新生血管的形成情况。高氧模型组和正常对照组生后19d幼鼠3只，行HE染色，光学显微镜下观察视网膜血管形态，观察突破内界膜的内皮细胞核数。取生后19d高氧组和对照组小鼠，分别取其视网膜组织并提取总RNA，反转录成cDNA后行反转录PCR，2％琼脂糖凝胶电泳并照相。提取视网膜总RNA，反转录成cDNA后（同RT-PCR），配制荧光定量实时PCR反应体系（总计20μl），在60℃检测荧光信号，分析图像。分别取高氧模型组和正常对照组P19小鼠行眼球切片，常规处理后TERT抗体孵育37℃60min，HRP酶标二抗孵育30min，DAB显色，中性胶封片，镜下观察并照相。结果高氧诱导模型小鼠生后12d眼底后极部出现大片无灌注区，生后14d眼底后极部出现新生血管迂曲、渗漏等视网膜血管病变。生后17—19d视网膜新生血管形成达到高峰。正常小鼠视网膜组织切片HE染色基本看不到突出内界膜的血管芽及血管管腔，内界膜下视网膜内的血管内皮细胞核散在分布、数量较少；高氧组见大量突出内界膜伸向玻璃体腔的血管管腔及血管芽，内界膜下视网膜内也有大量血管内皮细胞增生。19d高氧模型组小鼠视网膜TERT及bFGFmRNA表达较同日龄正常对照组小鼠明显提高，二者差异有统计学意义（F=8．575，5．667；P〈0．05）。生后19d实时PCR检测高氧模型组小鼠视网膜TERTmRNA表达较同日龄正常对照组小鼠明显上调，差异有统计学意义（F=173．104，P〈0．05）。生后19d高氧诱导小鼠视网膜新生血管模型中视网膜新生血管TERT表达阳性，同日龄对照组新生小鼠视网膜血管TERT表达阴性。结论高氧诱导视网膜新生血管小鼠模型中端粒酶逆转录酶和新生血管形成相关因子表达水平明显上调，可能会成为视网膜新生血管疾病预防和治疗的新靶点。
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 candalis 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 μm serial slices, hematoxylin-eosin staining, select one section every 60 μm 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 20μl ） was made. The Fluorescent signals were detected at 60 ℃. The expression of mTERT were confirmed by immunohistochemistry. At P19, 4 μm 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 ℃. 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 centrel 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 hyperxia 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 hyperxia 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.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2009年第3期199-205,共7页
Chinese Journal of Ophthalmology
基金
山东省自主创新重大科技专项计划基金（2006GG1102020）
山东省医学科学院课题（2007-29）
