MMP-2与MMP-9在糖尿病大鼠视网膜中的表达研究

发布时间：2018-01-29 17:12

  本文关键词： 糖尿病视网膜病变 基质金属蛋白酶2 基质金属蛋白酶9 大鼠　出处：《郑州大学》2011年硕士论文　论文类型：学位论文

【摘要】：糖尿病视网膜病变(diabetic retinopathy, DR)是糖尿病患者的严重并发症之一,是导致失明的重要原因,但DR的发病机制仍在探讨中。DR的严重程度与糖尿病病程呈正相关。在对发病机制的研究中,视网膜毛细血管周细胞损害,破坏了视网膜的内屏障,随后产生了各种血管生成因子和细胞外基质,促成了新生血管的形成。而增生期糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)的发生与新生血管的形成有关。近年来,国内外的大量研究针对于PDR的发病机制,但主要都集中在对血管生成因子的研究上面,而在对细胞外基质(extra cellularmatrix, ECM)在PDR的发病中所发挥的作用研究甚少。相关的研究表明,基质金属蛋白酶(matrixmetalloproteinases, MMPs)中MMP-2、MMP-9与新生血管的形成密切相关。 本实验拟建立糖尿病眼动物模型,应用免疫组化法测定视网膜中MMP-2、MMP-9蛋白含量的表达,初步探讨糖尿病病程的发展对MMP-2、MMP-9蛋白表达的影响。 材料和方法 选6周龄、健康、清洁级SD大鼠60只,雌雄不限,体重70-90g,常规母乳喂养,排除先天性近视及其它眼疾,随机分为4组。对照组(Ⅰ组)15只,腹腔注射等量空白缓冲液,饲养21周,其余3组均拟建糖尿病动物模型组,每组15只共45只,禁食水12小时后,按65mg/kg剂量连续3周左下腹腔小剂量多次注射链脲佐菌素(streptozocin,STZ),制作糖尿病大鼠模型。3天后测空腹血糖,血糖"g=16.5mmol/L以上,尿量及饮水明显增多者为糖尿病大鼠。其中Ⅱ组饲养13周,Ⅲ组饲养17周,Ⅳ组饲养21周,实验结束后,分别处死Ⅱ、Ⅲ、Ⅳ及Ⅰ组大鼠,取眼球行视网膜切片HE染色、免疫组织化学染色,通过与电脑相连的Biosens Digital Imaging system V1.6高清晰度彩色病理图文分析系统作细胞MMP-2蛋白、MMP-9蛋白表达的半定量分析,用平均光密度值来表示免疫反应表达程度。 结果 1.体重及空腹血糖变化比较：Ⅰ组体重明显高于Ⅱ组、Ⅲ组及Ⅳ组,其差异具有统计学意义(P0.05)；Ⅱ组、Ⅲ组及Ⅳ组体重两两相比无统计学意义。Ⅱ组、Ⅲ组及Ⅳ组空腹血糖明显高于Ⅰ组,其差异具有统计学意义(P0.05)；Ⅱ组、Ⅲ组与Ⅳ组空腹血糖两两相比无统计学意义。 2.免疫组化染色：MMP-2、MMP-9阳性细胞表现为胞浆着色呈棕黄色,主要在神经节细胞层胞浆和血管内皮细胞有阳性表达,糖尿病组大鼠随着病情的发展,视网膜内核层细胞有阳性表达。Ⅱ组、Ⅲ组及Ⅳ组阳性表达均高于Ⅰ组,差异有统计学意义(P0.05)；Ⅱ组、Ⅲ组及Ⅳ组之间比较,差异均有统计学意义(P0.05)。 结论 1.STZ小剂量多次腹腔注射是一种可靠的制备糖尿病眼动物模型的方法。 2.MMP-2、MMP-9在糖尿病视网膜上表达呈阳性,并随着糖尿病病程的发展,二者的表达呈递增状态。
[Abstract]:Diabetic retinopathy (DRR) is one of the serious complications of diabetic patients and an important cause of blindness. However, the pathogenesis of Dr is still under discussion. The severity of Dr is positively correlated with the course of diabetes. In the study of pathogenesis, retinal capillary pericytes damage and damage to the internal barrier of the retina. Various angiogenic factors and extracellular matrix were subsequently produced. And proliferative diabetic retinopathy in proliferative diabetic retinopathy. In recent years, a large number of studies at home and abroad focused on the pathogenesis of PDR, but mainly focused on the study of angiogenic factors. However, the role of extra cellular matrix (ECM) in the pathogenesis of PDR is very little. MMP-9 in matrix metalloproteinases (MMPs) is closely related to angiogenesis. In order to establish an animal model of diabetic eyes, the expression of MMP-2 and MMP-9 in retina was determined by immunohistochemical method, and the relationship between the development of diabetes mellitus and MMP-2 was preliminarily discussed. Effect of MMP-9 protein expression. Materials and methods Sixty healthy, clean SD rats of 6 weeks of age, male and female, weight 70-90 g, were selected. Routine breast-feeding was performed to rule out congenital myopia and other eye diseases. The rats were randomly divided into 4 groups: control group (group 鈪，

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