Rhodopsin和S-opsin在MNU诱导的大鼠视网膜损伤过程中的表达变化

目的:观察在N-甲基-N亚硝酸脲(MNU)诱导的大鼠视网膜外核层细胞损伤过程中视紫质(rhodopsin)和蓝光视蛋白(blue-sensitive opsin/S-opsin)在视网膜中的表达变化,分析其与MNU诱导的视网膜损伤的关系。方法:将30只SPF级50 d龄雌性SD大鼠随机分为正常对照组和MNU模型1、3、7、10 d组,每组各6只大鼠。模型组以腹腔注射MNU(40 mg/kg)建立MNU模型;正常对照组大鼠腹腔注射生理盐水(5 mL/kg)。右眼冰冻切片行苏木素-伊红(HE)染色判断视网膜损伤程度。通过逆转录-聚合酶链反应(RT-PCR)和免疫荧光检测各组视网膜中rhodopsin和S-opsin的mRNA及蛋白表达情况。结果:病理检测确定造模结果与以往实验一致,各组病理分级之间的差异显著(2=16.838,P0.01)。RT-PCR检测结果表明,与正常对照组相比,各MNU模型组rhodopsin和S-opsin的mRNA表达水平随MNU作用时间增加而逐渐降低且差异均显著(rhodopsin 1 d组P0.05;S-opsin 1 d组P0.01;3、7、10 d组rhodopsin和S-opsin均P0.01)。免疫荧光检测结果显示,在正常大鼠视网膜rhodopsin主要在光感受器细胞外段表达,MNU作用后rhodopsin主要在外核层表达,少量在内核层表达,并随MNU作用时间增加而逐渐表达降低。S-opsin在正常视网膜各层均有表达,在各模型组随MNU作用时间增加S-opsin的表达逐渐降低,在外核层和光感受器细胞内、外段尤为明显。结论:MNU诱导的视网膜外核层细胞损伤可降低rhodopsin和S-opsin的表达,并与MNU选择性引起光感受器细胞丧失有关。

Changes of rhodopsin and S-opsin expression in MNU-induced retinal damage in rats

AIM: To investigate the expression of rhodopsin and S-opsin/blue-sensitive opsin in damaged retina induced by N-methyl-N-nitrosourea (MNU). METHODS: Single intraperitoneal injection of MNU 40 mg/kg was given to twenty-four 50-day-old female Sprague-Dawley rats and the rats were examined sequentially on day 1, 3, 7 and 10 after MNU treatment (6 rats were randomly sacrificed at each time point). As control, six rats were injected with saline (5 mL/kg) and sacrificed on day 3 after injection. Pathological changes in retina were graded by the scores of HE staining. Expression of rhodopsin and S-opsin was detected by RT-PCR and immunofluorescence assay. RESULTS: The damaged retinas of rats were confirmed by pathological examination and the differences of pathological scores between different groups were statistical significant (²=16.838,P<0.01). RT-PCR results indicated that rhodopsin and S-opsin mRNA levels in retina decreased after MNU injection compared with normal control (rhodopsin on day 1,P<0.05;S-opsin on day 1 P<0.01; rhodopsin and S-opsin on day 3,7 and 10, P<0.01). Immunofluorescence assay demonstrated that in normal retina rhodopsin immunolabeling was mainly in photoreceptor outer segments. After MNU treatment the positive staining of rhodopsin was detected mainly in the outer nuclear layer with only a few staining in the inner nuclear layer, and the level of staining decreased with the increasing intervals after MNU injection. Moreover,the S-opsin immunolabeling emerged in all layers of retina. After MNU injection, the S-opsin immunolabeling levels decreased, especially in the photoreceptor cell inner and outer segments and the outer nuclear layer. CONCLUSION: In vivo MNU treatment induces a gradual decrease of rhodopsin and S-opsin expression in rat retina and relates to the apoptosis of retinal photoreceptor cells.