一种新型糖尿病视网膜病变大鼠模型的建立及其病理特点研究

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目的:探讨一种新型的糖尿病视网膜病变(DR)大鼠模型的建立及其病理特点。方法:取褐色挪威(BN)大鼠36只,分为对照组、链脲佐菌素(STZ)组和STZ+亚精胺(SP)组,每组12只。STZ组和STZ+SP组尾静脉注射STZ,STZ+SP组玻璃体注射亚精胺,对照组给与同体积的溶剂。造模后继续饲喂12周,期间监测体重和空腹血糖。利用超声成像系统观察视网膜中央动脉血流供应情况。动物处死后取眼球行苏木精-伊红染色,观察视网膜基本结构,分析各层厚度。视网膜消化铺片行过碘酸雪夫染色和神经胶质抗原2免疫荧光染色,观察微血管和周细胞情况。采用流式细胞术检测视网膜组织活性氧簇(ROS)水平;最后利用分子生物学技术检测紧密连接蛋白闭合蛋白-1(claudin-1)和咬合蛋白(occludin) mRNA和蛋白表达。多组重复测量的计量资料的比较采用重复测量的方差分析,组间两两比较采用LSD-n t法。n 结果:与对照组比较,STZ组和STZ+SP组大鼠体重下降(n P<0.05),血糖升高(n P<0.05);与STZ组比较,STZ+SP组大鼠视网膜中央动脉舒张末期血流速度和收缩末期血流速度下降(n P<0.05),搏动指数和阻力指数则升高(n P<0.05)。病理学观察可见,STZ组和STZ+SP组大鼠神经细胞核层区排列疏松、紊乱,视网膜丛区水肿,且STZ+SP组表现的更为严重。与STZ组比较,STZ+SP组视网膜厚度和外丛层厚度/视网膜总厚度增加(n P<0.05),毛细血管无细胞新生毛细血管形成增多和周细胞缺失明显。与对照组和STZ组比较,STZ+SP组视网膜组织ROS水平均升高(n P<0.05)。与对照组比较,STZ+SP组claudin-1和occludin mRNA下降(n P<0.05),相应的蛋白表达亦降低(n P<0.05)。n 结论:BN大鼠尾静脉注射STZ联合玻璃体注射SP能建立一种严重的DR动物模型,病变特征主要表现为更严重的视网膜血管顺应性下降,血流供应减少,视网膜水肿,新生无细胞毛细血管大量生成以及视网膜周细胞凋亡增多。“,”Objective:To explore the establishment of a novel rat model of diabetic retinopathy (DR) and its pathological characteristics.Methods:Thirty-six Brown Norway (BN) rats were divided into the control group (n n=12), streptozocin (STZ) group (n n=12) and STZ+spermidine (SP) group (n n=12). STZ group and STZ+SP group received tail vein injection of STZ. STZ+SP group received vitreous injection of SP, and the control group received vitreous injection of the same volume of solvent. The rats continued to feed for 12 weeks after modeling, during which weight and fasting blood glucose were monitored. The ultrasound imaging system was used to image the central retinal artery to observe the retinal blood supply. After sacrificed, the eyes were enucleated for hematoxylin-eosin staining to observe the basic structure of the retina, to analyze the thickness of each layer. Retina digestive preparations were for periodic acid-schiff staining and glial antigen 2 immunofluorescent staining to observe the microvessels and pericytes, respectively. Retinal reactive oxygen species (ROS) levels were observed by flow cytometry. Finally, the claudin-1 and occludin mRNA expressions in the retina tissue were detected by real-time polymerase chain reaction and the protein expressions were assessed by automatic protein quantification technology. Repeated measurements analysis of variance was used to compare among multiple groups, and LSD-n t method was used to compare between two groups.n Results:Compared with the control group, the STZ group and the STZ+SP group had significantly decreased body weight (n P<0.05) and increased blood glucose (n P<0.05) during the experiment. Compared with the STZ group, peak systolic velocity and end diastolic velocity of the central retinal artery in the STZ+SP group were markedly reduced (n P<0.05), while pulsatility index and resistance index were obviously raised (n P<0.05), the differences were statistically significant. Pathological observations showed that the arrangement of the neuronuclear layer was loose and disordered, and the retinal plexus area was edema in the STZ group and the STZ+SP group, moreover, the STZ+SP group performed more seriously. Compared with the STZ group, the thickness of the retina and the thickness of the outer plexus/total thickness of the retina were increased significantly in the STZ+SP group (n P<0.05), and the loss of capillary pericytes and the formation of acellular capillaries were also distinctly increased. Compared with the control group and the STZ group, ROS levels in the STZ+SP group were increased significantly (n P<0.05). Compared with the control group, the expressions of claudin-1 and occludin mRNA in the retina of the STZ+SP group were decreased significantly (n P<0.05), as well as the corresponding protein expressions (n P<0.05), the differences were statistically significant.n Conclusions:A severe animal model of DR lesions is established via tail vein injection of STZ combined with vitreous injection of SP in BN rats. The lesions are characterized by more severe decrease of retinal vascular compliance, reduction of blood flow supply, retinal edema, formation of a large number of new acellular capillaries and the increased apoptosis of retinal capillary pericytes.
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