Tenomodulin抑制氧诱导视网膜病变小鼠模型新生血管形成的研究

Anti-angiogenic effect of the transmemberane protein(Tenomodulin) on retinal neovascularization in a mouse model

目的探讨Tenom odulin(TeM)蛋白对C57BL/6J小鼠早产儿视网膜病变模型(ROP)视网膜新生血管形成的抑制作用。方法将60只7d龄C57BL/6J幼鼠随机分为高氧组(ROP组)、正常对照组各15只和TeM组30只。将ROP组小鼠置于体积分数(75%±2%)的高氧环境中5d,随后回到正常氧环境中饲养5d;对照组小鼠饲养在正常氧环境中;TeM组是将鼠1只眼玻璃体腔内注射1μgTeM,对侧眼注射PBS作为对照,然后置于体积分数(75%±2%)的高氧环境中饲养5d,之后返回正常氧环境中。17d时处死全部小鼠,收集各组眼球。视网膜荧光素灌注铺片观察视网膜血管的改变、计算视网膜无灌注区的面积;计数突破内界膜的内皮细胞数反映视网膜血管增生情况,观察TeM蛋白对视网膜新生血管形成的抑制作用及对视网膜可能的毒性反应;Westernblot检测TeM蛋白在视网膜内的表达。结果与高氧组(2.94±0.55)mm2及TeM组中对侧眼注射PBS(2.83±0.46)mm2的视网膜铺片中央非灌注区面积(mm2)相比,注射TeM的视网膜铺片(0.44±0.26)mm2,其中央非灌注区面积差异有统计学意义(P<0.01);每个视网膜切面突破内界膜的内皮细胞核数(10.57±2.95)与前二者(44.93±6.78、41.07±7.31)比较差异均有统计学意义(P<0.01)。注射TeM的视网膜冰冻切片未见炎症反应和细胞毒性破坏。Westernblot检测结果显示注射TeM的视网膜呈阳性表达,注射PBS的视网膜未出现条带反应,TeM的相对分子质量约为16000。结论TeM可有效抑制视网膜新生血管的形成,预示着TeM在预防和治疗眼部新生血管方面具有潜在的作用。

Background Retinal vascular diseases is a main causing-blindness eye disease. Tenomodulin（TeM） is proved to be an inhibitor of neovascularization. But its mechanism is still below understanding. Objective The present paper is to explore the anti-angiogenic effect of TeM protein on retinal neovascularization in a mouse model of retinopathy of prematurity （ROP）. Methods Sixty 7-day-old C57BL/6J mice were used in this study. Fifteen mice were exposed to（75 ±2）% oxygen for 5 days followed by 5 days in room air to create ROP models. Fifteen mice were raised in room air environment from birth through postnatal day 17 as normal groups,1μg TeN was intravitreally injected in lateral eyes and PBS was injected in the fellow eyes and then exposed to（75 ± 2）% oxygen in 30 mice. Eyeballs were obtained at postnatal day 17. Retinal blood vessel patterns were visualized by fluorescein angiography. The number of neovascular nuclei breaking the inner limiting membrane（ILM） was counted with a masked approach,and the inhibitory effects of TeN on retinal neovaseularization was observed. The morphology of retina was examined under the light microscope to evaluate the possible toxicity of TeM protein on retina. Western blot analysis was performed to detect the expression of TeM protein in retinal tissue. Results The central retinal nonperfusion area in ROP mouse was （2. 94± 0. 55 ） mm^2 and that of PBS-injected mouse was （2. 83 ± 0.46 ）mm^2 , showing a signifieant difference in comparison with TeM-injected mouse（0.44 ± 0.26 mm^2 ） （ P 〈 0.01 ）. The number of endothelial cells nuclei of new blood vessels breaking through inner limiting membrane（ILM） on each retinal cross-section was（ 10.57± 2.95 ）mm^2 in TeM-injected mouse, showing a significant different with that of ROP eye （ 44.93 ± 6.78 ） mm^2 and PBS-injected eye （ 41.07 ±7.31 ） mm^2 （ P 〈 0.01 ）. No obvious alteration of morphology in retinal section was found in TeM injected eye. Western blot assay showed an immunoreactive band of TeM with the molecular weight of 16 000. No positive expression was detected in PBS injected eye. Conclusion TeM can effectively inhibit pathological angiogenesis in C57BL/6J mouse. This result indicates a potential role of TeM in preventing and treating ocular neovascularization.