【摘要】： 目的:糖皮質激素廣泛應用于臨床疾病的治療。長期全身、局部應用糖皮質激素可導致激素性白內障,其發(fā)病機制尚不明確。目前已證實晶狀體上皮細胞存在糖皮質激素受體。糖皮質激素作用于人晶狀體上皮細胞后,與其受體結合,其激素受體復合物與相應靶基因的糖皮質激素反應元件(GRE)結合后調控大量靶基因的轉錄,進而影響相應靶基因的功能。其中主要涉及細胞膜的轉運和細胞骨架結構的改變等。晶狀體通過Na+,K+-ATP酶維持離子進出細胞的平衡,其功能的改變將會引起晶狀體細胞內水鈉潴留,形成白內障。波形蛋白是重要的細胞骨架蛋白,在晶狀體上皮細胞中適量表達,對于維持晶狀體細胞正常的形態(tài)和功能,具有重要意義。本實驗將應用糖皮質激素受體拮抗劑RU486探討糖皮質激素受體介導的大鼠激素性白內障Na+,K+-ATP酶和波形蛋白的變化,探討糖皮質激素受體在激素性白內障的發(fā)病中的作用機制。 方法:大鼠透明晶狀體隨機分為對照組、糖皮質激素誘導的激素白內障組(地塞米松5μM)、糖皮質激素受體拮抗劑RU486組(地塞米松5μM+RU486 5μM)。離體晶狀體孵育7天,倒置顯微鏡動態(tài)觀察晶狀體的透明度。HE染色分析三組晶狀體的組織形態(tài)學變化,分光光度計動態(tài)分析Na+,K+-ATP酶的活性,蛋白質印跡法和免疫組化法分析Na+,K+-ATP酶α1和波形蛋白的蛋白表達,RT-PCR分析Na+,K+-ATP酶α1和波形蛋白的mRNA表達。 結果:白內障組與其它兩組相比,晶狀體在第5天出現(xiàn)霧狀混濁(P0.05),第7天霧狀混濁更明顯(P0.01),而對照組和拮抗劑組保持透明。白內障組與其它兩組相比,Na+,K+-ATP酶活性隨孵育時間的延長而逐漸下降(P0.001),而對照組和拮抗劑組無明顯變化。HE染色顯示白內障組與對照組和拮抗劑組相比,晶狀體纖維結構排列紊亂,細胞間隙增寬。白內障組Na+,K+-ATP酶α1蛋白和mRNA表達下降,而對照組和拮抗劑組表達正常。白內障組波形蛋白的蛋白表達下降,而對照組和拮抗劑組表達正常。三組中波形蛋白mRNA表達無明顯改變。 結論:本研究表明糖皮質激素受體介導的晶狀體Na+,K+-ATP酶α1和酶活性以及波形蛋白的改變參與了激素性白內障的形成,并且發(fā)揮著重要作用。
[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 偽 1 and vimentin was detected by Western blot and immunohistochemistry, and mRNA expression of Na, K ATP enzyme 偽 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 偽 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 偽 1, enzyme activity and vimentin play an important role in the formation of hormonal cataract.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R776.1

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