脯氨酸羟化酶2对氧诱导视网膜病变形成的影响

发布时间：2018-07-31 15:20

【摘要】：研究背景:视网膜新生血管(retinal neovascularization, RNV)是一系列缺血缺氧性视网膜疾病的共同病理表现,包括:糖尿病视网膜病变(diabetic retinopathy, DR)、视网膜静脉阻塞(retinal vein occlusion, RVO)、早产儿视网膜病变(retinopathy of prematurity, ROP)等。由于视网膜缺氧,造成促血管生长因子如血管内皮生长因子(vascular endothelial growth factor , VEGF)表达量增加,从而促进视网膜血管内皮细胞分裂、增生,形成新生血管。目前,对于此类疾病的药物治疗主要是抑制VEGF从而抑制新生血管的生长,然而促血管生长因子种类繁多只针对VEGF的治疗效果并不理想。因此非常有必要在促血管内皮生长因子的上游寻找更有效的方法来治疗视网膜新生血管疾病。脯氨酸羟化酶2(proline hydroxylase 2, PHD2)是缺氧诱导因子(hypoxia-inducible factor, HIF)-1a的降解酶,而HIF-1a可以调节VEGF、EPO等多种因子的表达[1,2]。近年来,对于PHD2的新认识表明抑制其表达后具有促进肿瘤新生血管正常化的作用[3,4]。那么其对于眼部新生血管是否具有抑制作用尚未见报道。因此,本研究拟通过抑制PHD2,以观察其对视网膜新生血管的治疗效果。目的:PHD2对氧致小鼠视网膜病变(OIR)动物模型新生血管形成的影响。方法:40只7d龄的C57BL/6J幼鼠随机分为4组,正常对照组、高氧组及300mg/kg、500mg/kg PHD2抑制剂处理组。后3组放入动物氧舱,在(75±2) %的高氧环境中连续饲养5d,正常对照组于正常空气环境中饲养。处理组在结束高浓度给氧后,立即给予腹腔注射不同剂量的PHD2抑制剂(黄芩素),每天1次,连续5d,高氧组和正常对照组给予生理盐水腹腔注射。于出生第17d时处死小鼠,行Western blot和视网膜铺片, H-E染色后计数突破内界膜的内皮细胞核数目,免疫组化检测视网膜a-SMA和VEGF蛋白的表达。结果:Western blot显示视网膜存在PHD2表达,且高氧组蛋白表达量明显高于正常对照组(P0.05)。视网膜铺片显示高氧组中央部视网膜有毛细血管无灌注区,视网膜新生血管形成,动静脉均迂曲、扩张; 300mg/kg及500mg/kg PHD2抑制剂处理组较高氧组明显改善。突破视网膜内界膜的内皮细胞核数目,正常对照组、高氧组、300mg/kg和500mg/kgPHD2处理组分别为0.6±0.84、39.4±5.17、15.8±1.93和6.3±1.57个,组间比较差异有统计学意义(P0.05),高氧组最多,正常对照组最少。免疫组化显示300mg/kg和500mg/kgPHD2抑制剂处理组a-SMA表达较高氧组明显增加;高氧组、300mg/kg及500mg/kgPHD2抑制剂处理组的VEGF表达较正常对照组明显增强, 300mg/kg和500mg/kgPHD2抑制剂处理组与高氧组比较,VEGF表达量无明显降低。结论:视视网膜存在PHD2表达,抑制其表达后能有效改善缺氧所致小鼠视网膜血管异常,提示PHD2可能在其发病机制中具有一定的作用。
[Abstract]:Background: retinal neovascularization (retinal neovascularization, RNV) is a common pathological feature of a series of ischemic and hypoxic retinopathy, including diabetic retinopathy (diabetic retinopathy, DR), retinal vein occlusion (diabetic retinopathy, DR), retinal vein occlusion (retinal vein occlusion, RVO), retinopathy (retinopathy of prematurity, ROP) in premature infants and so on. Because of retinal hypoxia, the expression of vascular growth factors such as vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is increased, thus promoting the division, proliferation and angiogenesis of retinal vascular endothelial cells. At present, the main drug therapy for these diseases is to inhibit the growth of new blood vessels by inhibiting VEGF. However, the therapeutic effect of many kinds of promoting factors for VEGF is not ideal. Therefore, it is necessary to find a more effective way to treat retinal neovascularization disease upstream of vascular endothelial growth factor. Proline hydroxylase 2 (PHD2) is an anoxic inducible factor (hypoxia-inducible factor, HIF) 1a) degrading enzyme, while HIF-1a can regulate the expression of many factors such as VEGF PHD2. In recent years, new knowledge of PHD2 has shown that inhibiting its expression can promote the normalization of tumor neovascularization. Whether it has inhibitory effect on ocular neovascularization has not been reported. Therefore, the aim of this study was to observe the therapeutic effect of PHD 2 on retinal neovascularization. Objective to investigate the effect of 1: PHD 2 on angiogenesis in (OIR) animal model induced by oxygen. Methods 40 7 day old C57BL/6J rats were randomly divided into 4 groups: normal control group, hyperoxia group and 300mg / kg / kg PHD2 inhibitor group. The latter three groups were fed in (75 卤2)% hyperoxia environment for 5 days, while the control group was fed in normal air environment. The treatment group was treated with different doses of PHD2 inhibitor (baicalein) once a day for 5 days. The hyperoxia group and the normal control group were intraperitoneally injected with normal saline. On the 17th day of birth, the mice were killed, Western blot and retina preparation were performed, the number of endothelial nuclei breaking through the inner boundary membrane was counted after H-E staining, and the expression of a-SMA and VEGF protein in retina was detected by immunohistochemistry. Results blot showed that there was PHD2 expression in the retina, and the expression of hyperoxia histone was significantly higher than that in the normal control group (P0.05). Retinal smears showed that there was no capillary perfusion area in the central retina of hyperoxia group, retinal neovascularization, arteriovenous detour and dilatation, and 300mg/kg and 500mg/kg PHD2 inhibitor treatment group were significantly improved compared with hyperoxia group. The number of endothelial nuclei breaking through the inner membrane of retina was 0.6 卤0.84g / kg in normal control group, 39.4 卤5.17g / kg in hyperoxia group and 6.3 卤1.57 in 500mg/kgPHD2 treatment group, respectively. There was significant difference between the two groups (P0.05), the hyperoxia group was the most, and the normal control group was the least. Immunohistochemistry showed that a-SMA expression in 300mg/kg and 500mg/kgPHD2 inhibitor groups was significantly higher than that in hyperoxia group. The expression of VEGF in the hyperoxia group (300mg / kg) and in the 500mg/kgPHD2 inhibitor group was significantly higher than that in the normal control group. The expression of VEGF in the 300mg/kg and 500mg/kgPHD2 inhibitor groups was not significantly lower than that in the hyperoxia group. Conclusion: there is PHD2 expression in the retina, which can effectively ameliorate the retinal vascular abnormalities induced by hypoxia, suggesting that PHD2 may play a role in the pathogenesis of retinopathy.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1

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