视网膜新生血管形成机制与防治的实验研究

发布时间：2019-03-15 12:00

【摘要】： [目的]：研究氧诱导的视网膜新生血管形成的机制及防治方法。 [方法]：20只新生C57BL/6J小鼠随机分为对照组和实验组,各10只。实验组P7置75%氧浓度环境中,P12返回正常空气中,对照组始终置于正常空气环境中,P17行荧光素灌注造影、视网膜铺片及病理切片,观察视网膜新生血管生成情况；幼鼠P12、P14、P17对照组和实验组各6只,取视网膜,应用realtime-PCR定量检测各时间点Annexin A2mRNA和TPAmRNA水平；HSS治疗组10只OIR模型小鼠腹腔注射饱和氢盐水、HSS对照组10只OIR模型小鼠腹腔注射生理盐水,正常组10只小鼠分别于P17用高分子量荧光素灌注造影观察血管分布与形态,HE染色计数突破内界膜的血管内皮细胞核数,用realtime-PCR和免疫组化检测视网膜VEGF表达,并测定丙二醛含量。 [结果]：1.荧光素灌注造影结果显示实验组小鼠视网膜中央呈无灌注区域,视网膜血管迂曲扩张、荧光素渗漏,对照组小鼠视网膜血管分布均匀,未见无灌注区和荧光素渗漏；病理切片可见实验组小鼠突破内界膜的血管内皮细胞核数为79.70±7.57,对照组为0.28±0.12,两组具有显著差异(P0.01)。2.P12和P17时,实验组与对照组Annexin A2mRNA和TPA mRNA的表达量无显著差异(P0.05)；P14时,实验组Annexin A2mRNA和TPA mRNA表达量均比对照组高,且具有显著差异(P0.01)。3.HSS治疗组荧光素灌注造影未见无灌注区、新生血管丛及荧光素渗漏；HSS对照组荧光素灌注造影视网膜视乳头周边可见大片无灌注区,视网膜血管不规则扩、迂曲,无灌注区周围可见大量新生血管丛,伴明显荧光素渗漏；正常组荧光素灌注造影视网膜未见无灌注区及荧光素渗漏。病理切片结果显示HSS治疗组、HSS对照组和正常组突破内界膜的血管内皮细胞核数分别为41.00±8.01、79.70±7.57和0.90±1.28,HSS治疗组与正常组无统计学差异(P0.05),HSS治疗组与HSS对照组具有显著差异(P0.01)；免疫组织化学实验显示VEGF表达于神经节细胞层、内核层和色素上皮细胞层,VEGF蛋白阳性表达HSS治疗组、HSS对照组和正常组分别为1.46±0.01、2.92±0.70和1.30±0.06；HSS治疗组、HSS对照组和正常组VEGFmRNA表达量分别为1.94±0.12、7.40±0.04和1.00±0.03,HSS治疗组与正常组之间无统计学意义(P0.05),HSS治疗组与HSS对照组有显著性差异(P0.01)；小鼠视网膜中MDA含量HSS治疗组为16.07±1.05 nmol/mg prot, HSS对照组为22.42±2.24 nmol/mg prot,正常组为5.17±4.23 nmol/mg prot, HSS治疗组与HSS对照组具有显著差异(P0.01),正常组与HSS对照组有显著差别(P0.01)。 [结论]：氧诱导视网膜新生血管小鼠模型稳定、可靠、重复性高,可做为研究视网膜新生血管疾病发病机制和防治方法的动物模型；Annexin A2和TPA与氧诱导视网膜新生血管的形成密切相关；饱和氢盐水对氧诱导的视网膜新生血管的形成有抑制作用。
[Abstract]:Objective: to study the mechanism, prevention and treatment of oxygen-induced retinal neovascularization. [methods]: 20 newborn C57BL/6J mice were randomly divided into control group (n = 10) and experimental group (n = 10). P7 was placed in 75% oxygen concentration in the experimental group, P12 returned to the normal air, and the control group was always in the normal air. P17 was performed fluorescein perfusion angiography, retinal preparation and pathological sections to observe the retinal neovascularization. The retina of P12, P14, P17 control group and experimental group were collected and the levels of Annexin A2mRNA and TPAmRNA were measured by realtime-PCR. In the HSS treatment group, 10 OIR mice were injected with saturated hydrogen saline intraperitoneally, and 10 OIR mice in the HSS control group were injected with saline intraperitoneally. In the normal group, 10 mice in the normal group were given high molecular weight fluorescein perfusion angiography to observe the distribution and morphology of the blood vessels. HE staining was used to count the nuclei of vascular endothelial cells which broke through the inner limiting membrane. The expression of VEGF in retina was detected by realtime-PCR and immunohistochemistry, and the content of malondialdehyde (MDA) was measured. [results]: 1. The results of fluorescein perfusion angiography showed that there was no perfusion area in the central retina of the experimental group, the retinal vessels were dilated roundly and fluorescein leakage was found in the experimental group, while the retinal vessels in the control group were evenly distributed, no perfusion area and no fluorescein leakage were found. Pathological sections showed that the number of endothelial cell nuclei breaking through the inner limiting membrane was 79.70 卤7.57 in the experimental group and 0.28 卤0.12 in the control group. There was significant difference between the two groups (P0.01). 2. P12 and P17 were significantly different from those in the control group (P < 0.01). There was no significant difference in the expression of Annexin A2mRNA and TPA mRNA between the experimental group and the control group (P0.05). At P14, the expression of Annexin A2mRNA and TPA mRNA in experimental group was higher than that in control group (P0.01). 3. There was no perfusion area, neovascularization and fluorescein leakage in fluorescein perfusion angiography in HSS group. In the HSS control group, large areas of no perfusion were seen around the optic papillae by fluorescein perfusion. The retinal vessels were dilated irregularly, and a large number of neovascularization and fluorescein leakage were seen around the no perfusion areas. In the normal group, no fluorescein leakage and no perfusion were found in the omentum. Pathological sections showed that the number of endothelial cell nuclei breaking through the inner boundary membrane in HSS treatment group, HSS control group and normal group were 41.00 卤8.01, 79.70 卤7.57 and 0.90 卤1.28, respectively. There was no significant difference between HSS treatment group and normal group (P0.05). There was significant difference between HSS treatment group and HSS control group (P0.01). Immunohistochemistry showed that VEGF was expressed in ganglion cell layer, inner nuclear layer and pigment epithelial layer. The positive expression of VEGF protein in HSS group, HSS control group and normal group were 1.46 卤0.01,2.92 卤0.70 and 1.30 卤0.06, respectively. The expression of VEGFmRNA in HSS treatment group, HSS control group and normal group was 1.94 卤0.12, 7.40 卤0.04 and 1.00 卤0.03, respectively. There was no significant difference between the two groups (P0.05). There was significant difference between HSS treatment group and HSS control group (P0.01). The content of MDA in the retina of mice was 16.07 卤1.05 nmol/mg prot, HSS in HSS group, 22.42 卤2.24 nmol/mg prot, in control group, 5.17 卤4.23 nmol/mg prot, HSS in normal group and 5.17 卤4.23 nmol/mg prot, HSS in HSS control group (P0.01). There was significant difference between normal group and HSS control group (P0.01). [conclusion]: oxygen-induced retinal neovascularization mouse model is stable, reliable and reproducible. It can be used as an animal model to study the pathogenesis, prevention and treatment of retinal neovascularization disease. Annexin A2 and TPA are closely related to the formation of retinal neovascularization induced by oxygen, and saturated hydrogen salt can inhibit the formation of oxygen-induced retinal neovascularization.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

【共引文献】