VEGF、nNOS在STZ诱导的糖尿病大鼠视网膜组织中表达变化的研究

发布时间：2018-04-18 08:20

本文选题：VEGF + nNOS　；参考：《昆明医学院》2011年硕士论文

【摘要】：目的：研究血管内皮生长因子(vascular endothelial growth factor, VEGF)、神经元型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)在早期糖尿病大鼠视网膜中的定位及表达变化,探讨二者在糖尿病视网膜病变(diabetic retinopathy, DR)发生发展中的作用,为DR的早期防治提供新的理论依据。方法：选取健康雄性SD大鼠46只,随机分成实验组和正常对照组。实验组用链脲佐菌素(streptozocin, STZ)大剂量(65mg/kg)一次性腹腔注射诱导1型糖尿病模型,对照组注射同样体积的柠檬酸缓冲液。造成糖尿病模型后按糖尿病病程分为三个实验组：3周组,6周组,9周组,每组10只。麻醉大鼠,固定眼球,石蜡包埋、切片,进行苏木素/伊红(Hematoxylin-Eosin, HE)染色,光镜下观察视网膜组织结构变化。免疫组织化学SP法,计算机图像分析技术及蛋白质免疫印迹技术检测视网膜中VEGF、nNOS蛋白表达水平及其分布。结果：HE染色显示,视网膜在糖尿病3周时变化不明显。随着病程的延长,病变不断加重,出现视网膜微血管玻璃样变、管壁增厚及管腔狭窄等变化。免疫组织化学检测显示,正常组VEGF阳性细胞主要分布于视网膜的神经节细胞层和内网状层。随着糖尿病病程的进展,VEGF表达的空间范围扩大到视网膜的内核层、外网状层及感光细胞层,VEGF蛋白表达水平逐渐升高,差异有统计学意义(P0.05)；nNOS主要分布于各组大鼠视网膜的神经节细胞及内网状层的双极细胞。nNOS免疫阳性反应在正常对照组和糖尿病3周组之间差异无统计学意义(P0.05),糖尿病6周和9周组nNOS阳性细胞数显著高于正常对照组,差异有统计学意义(P0.05),糖尿病不同病程组之间相比,差异有统计学意义(P0.05)。Western Blotting结果显示,VEGF蛋白条带在正常对照组表达较弱,与正常对照组相比,糖尿病3、6、9周组VEGF蛋白表达条带相对光密度值逐渐增加,差异有统计学意义(P0.05)；正常对照组和糖尿病组均见nNOS蛋白表达条带。糖尿病3周组nNOS蛋白表达条带相对光密度值与对照组相比,差异无统计学意义(P0.05),糖尿病6周及9周组nNOS蛋白表达条带相对光密度值显著高于正常对照组,差异有统计学意义(P0.05),糖尿病不同病程组之间相比,差异有统计学意义(P0.05)。相关分析结果显不,VEGF和nNOS蛋白表达水平存在着明显的正相关关系(P0.01)。结论：1 STZ诱导的1型糖尿病大鼠模型可以作为研究人类背景期DR的理想模型。 2在DR早期,随着糖尿病病程的进展,VEGF及nNOS蛋白表达逐渐升高,推测与视网膜三级神经元传导功能及神经细胞功能紊乱有关。 3 VEGF和nNOS在DR早期的发生发展过程中存在着明显的正相关关系。
[Abstract]:Objective: to study the localization and expression of vascular endothelial growth factor (VEGF) and neuronal nitric oxide synthase (nNOS) in the retina of early diabetic rats.To explore the role of both of them in the pathogenesis and development of diabetic retinopathy (DRD), and to provide a new theoretical basis for the early prevention and treatment of Dr.Methods: 46 healthy male SD rats were randomly divided into experimental group and normal control group.The model of type 1 diabetes was induced by intraperitoneal injection of streptozocin (STZ) at a large dose of 65 mg / kg in the experimental group and citric acid buffer of the same volume in the control group.According to the course of diabetes, the diabetic model was divided into 3 experimental groups: 3 weeks, 6 weeks and 9 weeks, 10 rats in each group.The rats were anesthetized, fixed eyeball, embedded in paraffin, sectioned and stained with hematoxylin / eosin (Hematoxylin-Eosin). The changes of retinal tissue structure were observed under light microscope.Immunohistochemical SP method, computer image analysis and Western blot technique were used to detect the expression and distribution of VEGF nNOS protein in retina.Results the changes of retina were not obvious at 3 weeks after diabetes.With the prolongation of the course of disease, the pathological changes were aggravated, such as retinal microvascular vitreous degeneration, wall thickening and lumen stenosis.Immunohistochemical examination showed that VEGF positive cells in normal group were mainly distributed in ganglion cell layer and inner reticular layer of retina.With the progression of diabetes mellitus, the expression of VEGF was extended to the inner layer of the retina, and the expression of VEGF protein increased gradually in the outer reticular layer and photoreceptor cell layer.The immunoreactivity of nNOS in the ganglion cells of the retina and the bipolar cells in the inner reticular layer was not significantly different between the normal control group and the diabetic 3-week group (P 0.05).The number of nNOS positive cells in the 6 and 9 week groups was significantly higher than that in the normal control group.There was a significant difference in the expression of VEGF-VEGF-protein in the normal control group and in the normal control group. The results of Western Blotting showed that the expression of VEGF-VEGF-protein was weaker in the normal control group than that in the normal control group.The relative optical density of VEGF protein expression band increased gradually in the control group and the diabetic group at 9 weeks, the difference was statistically significant (P 0.05), and the nNOS protein expression bands were found in the normal control group and the diabetic group.Compared with the control group, the relative optical density of nNOS protein expression band in the diabetes mellitus group was not significantly different from that in the control group (P 0.05). The relative optical density value of the nNOS protein expression band in the 6 and 9 weeks diabetic group was significantly higher than that in the normal control group.The difference was statistically significant (P 0.05), and there was significant difference between the different course groups of diabetes mellitus (P 0.05).The correlation analysis showed that there was a significant positive correlation between the expression level of nNOS and nNOS protein.Conclusion the rat model of type 1 diabetes induced by 1: 1 STZ can be used as an ideal model for the study of human background Dr.2 at the early stage of Dr, the expression of VEGF and nNOS protein increased gradually with the progression of diabetes mellitus, which was related to the conduction function of tertiary neurons in the retina and the dysfunction of nerve cell function.(3) there was a positive correlation between VEGF and nNOS in the early stage of Dr.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R587.2;R774.1

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