糖皮质激素受体介导的激素性白内障形成机制的研究
发布时间：2018-11-05 09:35
【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(P0.05),第7天雾状混浊更明显(P0.01),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(P0.001),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。
[Abstract]:Objective: glucocorticoids are widely used in the treatment of clinical diseases. Long-term systemic, local use of glucocorticoid can lead to hormone-induced cataract, its pathogenesis is unclear. The presence of glucocorticoid receptors in lens epithelial cells has been confirmed. Glucocorticoid acts on human lens epithelial cells and binds to its receptor. The hormone receptor complex binds to the glucocorticoid response element (GRE) of the corresponding target gene and regulates the transcription of a large number of target genes. Furthermore, the function of the target gene is affected. It mainly involves cell membrane transport and cytoskeleton structure change. Lens maintains the balance of ion entering and leaving cells through Na, K-ATP enzyme, and the change of its function will lead to the retention of water and sodium in lens cells and the formation of cataract. Vimentin is an important cytoskeleton protein which is expressed in lens epithelial cells and plays an important role in maintaining the normal morphology and function of lens cells. In this study, glucocorticoid receptor antagonist (RU486) was used to investigate the changes of Na, K-ATP enzyme and vimentin in glucocorticoid receptor-mediated rat steroid-induced cataract. To explore the role of glucocorticoid receptor in the pathogenesis of hormonal cataract. Methods: rat clear lens were randomly divided into control group and glucocorticoid-induced cataract group (dexamethasone 5 渭 M), glucocorticoid receptor antagonist RU486 group (dexamethasone 5 渭 M RU486 5 渭 M). After incubation for 7 days in vitro, the transparency of the lens was observed dynamically by inverted microscope. The histomorphological changes of the three groups were analyzed by HE staining and the activity of Na, K-ATP enzyme was dynamically analyzed by spectrophotometer. Protein expression of Na, K-ATP enzyme 伪 1 and vimentin was detected by Western blot and immunohistochemistry, and mRNA expression of Na, K ATP enzyme 伪 1 and vimentin by RT-PCR. Results: compared with the other two groups, the lens haze appeared on the 5th day (P0.05), and on the 7th day it was more obvious (P0.01), while the control group and the antagonist group remained transparent. Compared with the other two groups, the activity of, Na, K-ATP enzyme in cataract group decreased gradually with the increase of incubation time (P0. 001), but there was no significant change in control group and antagonist group. HE staining showed that the activity of, Na, K-ATP enzyme in cataract group was higher than that in control group and antagonist group. The structure of lens fibers was disordered and the intercellular space widened. The expression of Na, K-ATP 伪 1 protein and mRNA decreased in cataract group, but normal in control group and antagonist group. The expression of vimentin decreased in cataract group, but normal in control group and antagonist group. There was no significant change in vimentin mRNA expression in the three groups. Conclusion: this study suggests that glucocorticoid receptor mediated changes in Na, K-ATP 伪 1, enzyme activity and vimentin play an important role in the formation of hormonal cataract.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R776.1
本文编号：2311661
[Abstract]:Objective: glucocorticoids are widely used in the treatment of clinical diseases. Long-term systemic, local use of glucocorticoid can lead to hormone-induced cataract, its pathogenesis is unclear. The presence of glucocorticoid receptors in lens epithelial cells has been confirmed. Glucocorticoid acts on human lens epithelial cells and binds to its receptor. The hormone receptor complex binds to the glucocorticoid response element (GRE) of the corresponding target gene and regulates the transcription of a large number of target genes. Furthermore, the function of the target gene is affected. It mainly involves cell membrane transport and cytoskeleton structure change. Lens maintains the balance of ion entering and leaving cells through Na, K-ATP enzyme, and the change of its function will lead to the retention of water and sodium in lens cells and the formation of cataract. Vimentin is an important cytoskeleton protein which is expressed in lens epithelial cells and plays an important role in maintaining the normal morphology and function of lens cells. In this study, glucocorticoid receptor antagonist (RU486) was used to investigate the changes of Na, K-ATP enzyme and vimentin in glucocorticoid receptor-mediated rat steroid-induced cataract. To explore the role of glucocorticoid receptor in the pathogenesis of hormonal cataract. Methods: rat clear lens were randomly divided into control group and glucocorticoid-induced cataract group (dexamethasone 5 渭 M), glucocorticoid receptor antagonist RU486 group (dexamethasone 5 渭 M RU486 5 渭 M). After incubation for 7 days in vitro, the transparency of the lens was observed dynamically by inverted microscope. The histomorphological changes of the three groups were analyzed by HE staining and the activity of Na, K-ATP enzyme was dynamically analyzed by spectrophotometer. Protein expression of Na, K-ATP enzyme 伪 1 and vimentin was detected by Western blot and immunohistochemistry, and mRNA expression of Na, K ATP enzyme 伪 1 and vimentin by RT-PCR. Results: compared with the other two groups, the lens haze appeared on the 5th day (P0.05), and on the 7th day it was more obvious (P0.01), while the control group and the antagonist group remained transparent. Compared with the other two groups, the activity of, Na, K-ATP enzyme in cataract group decreased gradually with the increase of incubation time (P0. 001), but there was no significant change in control group and antagonist group. HE staining showed that the activity of, Na, K-ATP enzyme in cataract group was higher than that in control group and antagonist group. The structure of lens fibers was disordered and the intercellular space widened. The expression of Na, K-ATP 伪 1 protein and mRNA decreased in cataract group, but normal in control group and antagonist group. The expression of vimentin decreased in cataract group, but normal in control group and antagonist group. There was no significant change in vimentin mRNA expression in the three groups. Conclusion: this study suggests that glucocorticoid receptor mediated changes in Na, K-ATP 伪 1, enzyme activity and vimentin play an important role in the formation of hormonal cataract.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R776.1
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本文编号：2311661
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