【作者】 谢国丽；

【导师】 严宏；

【作者基本信息】 第四军医大学 ， 眼科学， 2010， 硕士

【摘要】 目的:糖皮质激素广泛应用于临床疾病的治疗。长期全身、局部应用糖皮质激素可导致激素性白内障,其发病机制尚不明确。目前已证实晶状体上皮细胞存在糖皮质激素受体。糖皮质激素作用于人晶状体上皮细胞后,与其受体结合,其激素受体复合物与相应靶基因的糖皮质激素反应元件(GRE)结合后调控大量靶基因的转录,进而影响相应靶基因的功能。其中主要涉及细胞膜的转运和细胞骨架结构的改变等。晶状体通过Na+,K+-ATP酶维持离子进出细胞的平衡,其功能的改变将会引起晶状体细胞内水钠潴留,形成白内障。波形蛋白是重要的细胞骨架蛋白,在晶状体上皮细胞中适量表达,对于维持晶状体细胞正常的形态和功能,具有重要意义。本实验将应用糖皮质激素受体拮抗剂RU486探讨糖皮质激素受体介导的大鼠激素性白内障Na+,K+-ATP酶和波形蛋白的变化,探讨糖皮质激素受体在激素性白内障的发病中的作用机制。方法:大鼠透明晶状体随机分为对照组、糖皮质激素诱导的激素白内障组(地塞米松5μM)、糖皮质激素受体拮抗剂RU486组(地塞米松5μM+RU486 5μM)。离体晶状体孵育7天,倒置显微镜动态观察晶状体的透明度。HE染色分析三组晶状体的组织形态学变化,分光光度计动态分析Na+,K+-ATP酶的活性,蛋白质印迹法和免疫组化法分析Na+,K+-ATP酶α1和波形蛋白的蛋白表达,RT-PCR分析Na+,K+-ATP酶α1和波形蛋白的mRNA表达。结果:白内障组与其它两组相比,晶状体在第5天出现雾状混浊(P<0.05),第7天雾状混浊更明显(P<0.01),而对照组和拮抗剂组保持透明。白内障组与其它两组相比,Na+,K+-ATP酶活性随孵育时间的延长而逐渐下降(P<0.001),而对照组和拮抗剂组无明显变化。HE染色显示白内障组与对照组和拮抗剂组相比,晶状体纤维结构排列紊乱,细胞间隙增宽。白内障组Na+,K+-ATP酶α1蛋白和mRNA表达下降,而对照组和拮抗剂组表达正常。白内障组波形蛋白的蛋白表达下降,而对照组和拮抗剂组表达正常。三组中波形蛋白mRNA表达无明显改变。结论:本研究表明糖皮质激素受体介导的晶状体Na+,K+-ATP酶α1和酶活性以及波形蛋白的改变参与了激素性白内障的形成,并且发挥着重要作用。更多还原

【Abstract】 PURPOSE: Cataract formation can be induced by prolonged use of glucocorticoids. The underlying mechanism is not fully understood yet. The presence of the functional glucocorticoid receptor (GR) in human and rat lens epithelial cells suggests that glucocorticoids target lens epithelial cells directly and specifically. Glucocorticoids exert their effects by binding to a specific intracellular receptor, the glucocorticoid receptor (GR), which acts as a ligand dependant transcription factor. The ligand-receptor complex dimerizes, translocates to the nucleus, and binds to a cis acting element, the glucocorticoid response element (GRE), to modulate the expression of target genes. The glucocorticoid–GR binding to a GRE located on that many target genes to directly modulate transcription, which indicate that a broad range of transcripts are altered. The main groupings of transcripts identified were involved in cell structure/extracellular matrix and adhesion, catalysis, transport protein activity, apoptosis, cell growth, and development and cell cycle. Na+, K+-ATPase has long been recognized for its role in regulating electrolyte concentration in the lens, contributing to lens transparency. Vimentin is an important cytoskeletal protein in the epithelial cells of the lens, which plays important roles in maintaining the normal morphology and function of the lens. It’s interesting to know whether the changes of Na+, K+-ATPase and vimentin can be induced through the specific GR activation in glucocorticord-induced cataract formation in rat lens in vitro.METHODS: Rat clear lenses were cultured in vitro and were treated with or without dexamethasone (Dex) or RU486(a glucocorticoid receptor antagonist). The lenses were cultured for 7 days and photographed daily to record the development of opacity. The changes of morphology were examined by HE staining. The activity of Na+, K+-ATPase was determined by using spectrophotometric analysis. The mRNA and protein expression of Na+, K+-ATPaseα1 and vimentin were examined by RT-PCR, and Western blot analysis and immunohistochemistry were conducted, respectively.RESULTS: Mist-like opacity of the lenses was observed as early as 5 days after incubation with dexamethasone (P<0.05). The opacity was more obvious at day 7 in the Dex group (P<0.01). At day 7, HE staining showed an orderly arrangement of fiber cells in the control group and the RU486 group. However, in the Dex group, the arrangement of fiber cells was disrupted and the lenses exhibited expanded extracellular lacunae. The activity of Na+, K+-ATPase in the only Dex-treated group decreased in a time-dependent manner. There was no significant loss of enzyme activity in either the control or the RU486 group throughout the incubation peroid (P<0.001). Both the protein and mRNA expression level of Na+, K+-ATPaseα1 decreased in the Dex-treated group. The expression of vimentin protein decreased in the Dex-treated group but its expression of mRNA maintained nomal.CONCLUSIONS: These results suggest that the GR-mediated reduction of Na+, K+-ATPase and vimentin may contrubite to the formation of steriod-induced cataract.更多还原