本文選題：VEGF + nNOS�。� 參考：《昆明醫(yī)學院》2011年碩士論文

【摘要】：目的：研究血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)、神經(jīng)元型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)在早期糖尿病大鼠視網(wǎng)膜中的定位及表達變化,探討二者在糖尿病視網(wǎng)膜病變(diabetic retinopathy, DR)發(fā)生發(fā)展中的作用,為DR的早期防治提供新的理論依據(jù)。 方法：選取健康雄性SD大鼠46只,隨機分成實驗組和正常對照組。實驗組用鏈脲佐菌素(streptozocin, STZ)大劑量(65mg/kg)一次性腹腔注射誘導1型糖尿病模型,對照組注射同樣體積的檸檬酸緩沖液。造成糖尿病模型后按糖尿病病程分為三個實驗組：3周組,6周組,9周組,每組10只。麻醉大鼠,固定眼球,石蠟包埋、切片,進行蘇木素/伊紅(Hematoxylin-Eosin, HE)染色,光鏡下觀察視網(wǎng)膜組織結(jié)構(gòu)變化。免疫組織化學SP法,計算機圖像分析技術(shù)及蛋白質(zhì)免疫印跡技術(shù)檢測視網(wǎng)膜中VEGF、nNOS蛋白表達水平及其分布。 結(jié)果：HE染色顯示,視網(wǎng)膜在糖尿病3周時變化不明顯。隨著病程的延長,病變不斷加重,出現(xiàn)視網(wǎng)膜微血管玻璃樣變、管壁增厚及管腔狹窄等變化。免疫組織化學檢測顯示,正常組VEGF陽性細胞主要分布于視網(wǎng)膜的神經(jīng)節(jié)細胞層和內(nèi)網(wǎng)狀層。隨著糖尿病病程的進展,VEGF表達的空間范圍擴大到視網(wǎng)膜的內(nèi)核層、外網(wǎng)狀層及感光細胞層,VEGF蛋白表達水平逐漸升高,差異有統(tǒng)計學意義(P0.05)；nNOS主要分布于各組大鼠視網(wǎng)膜的神經(jīng)節(jié)細胞及內(nèi)網(wǎng)狀層的雙極細胞。nNOS免疫陽性反應(yīng)在正常對照組和糖尿病3周組之間差異無統(tǒng)計學意義(P0.05),糖尿病6周和9周組nNOS陽性細胞數(shù)顯著高于正常對照組,差異有統(tǒng)計學意義(P0.05),糖尿病不同病程組之間相比,差異有統(tǒng)計學意義(P0.05)。Western Blotting結(jié)果顯示,VEGF蛋白條帶在正常對照組表達較弱,與正常對照組相比,糖尿病3、6、9周組VEGF蛋白表達條帶相對光密度值逐漸增加,差異有統(tǒng)計學意義(P0.05)；正常對照組和糖尿病組均見nNOS蛋白表達條帶。糖尿病3周組nNOS蛋白表達條帶相對光密度值與對照組相比,差異無統(tǒng)計學意義(P0.05),糖尿病6周及9周組nNOS蛋白表達條帶相對光密度值顯著高于正常對照組,差異有統(tǒng)計學意義(P0.05),糖尿病不同病程組之間相比,差異有統(tǒng)計學意義(P0.05)。相關(guān)分析結(jié)果顯不,VEGF和nNOS蛋白表達水平存在著明顯的正相關(guān)關(guān)系(P0.01)。 結(jié)論：1 STZ誘導的1型糖尿病大鼠模型可以作為研究人類背景期DR的理想模型。 2在DR早期,隨著糖尿病病程的進展,VEGF及nNOS蛋白表達逐漸升高,推測與視網(wǎng)膜三級神經(jīng)元傳導功能及神經(jīng)細胞功能紊亂有關(guān)。 3 VEGF和nNOS在DR早期的發(fā)生發(fā)展過程中存在著明顯的正相關(guān)關(guān)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.
【學位授予單位】：昆明醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R587.2;R774.1

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