Ozaki Taku, Nakazawa Mitsuru, Kudo Takashi, Hirano Satoshi, Suzuki Kaori, Ishiguro Sei-ichi
Department of Ophthalmology, Hirosaki University Graduate School of Medicine , Hirosaki , Japan and.
Curr Eye Res. 2014 Dec;39(12):1221-31. doi: 10.3109/02713683.2014.907430. Epub 2014 Jul 9.
RPE65, a retinal pigment epithelium-specific 65-kDa protein, plays a critical role in the visual cycle of the eye. Rpe65(-/-) mice develop vision loss due to a lack of 11-cis-retinal, degradation of M-opsin and mislocalization of S-opsin. Several studies have suggested that 9-cis-β-carotene, a precursor of 9-cis-retinal and all-trans-retinal, could have therapeutic applications in vision loss. We therefore examined whether Dunaliella bardawil, a 9-cis-β-carotene-rich alga, protects against the degradation of M-opsin using Rpe65(-/-) mouse retinal explant cultures.
The eyes of three-week-old Rpe65(-/-) and C57BL/6 J mice were enucleated, and the corneas were removed. The eyecups were incubated with culture medium in the absence or presence of D. bardawil for 6 h to 4 days. Localizations of M-opsin proteins in the retina were observed immunohistochemically. Expression levels of M-opsin, S-opsin and rhodopsin proteins were evaluated by Western blot analysis.
In C57BL/6 J mouse retina, no change was observed in localization and expression levels of M-opsin in the explant culture system. In Rpe65(-/-) mouse retina, the amount of M-opsin protein was decreased in the photoreceptor outer segment after 6 h to 4 days of culture. However, the presence of D. bardawil significantly ameliorated this decrease. In contrast, expression levels of S-opsin and rhodopsin were unchanged in the presence of the explant culture.
These results demonstrate that D. bardawil treatment protects against M-opsin degradation in Rpe65(-/-) mouse retina and suggest that D. bardawil has therapeutic potential for retinal degeneration caused by Rpe65 gene mutation, such as Leber congenital amaurosis and retinitis pigmentosa.
视网膜色素上皮特异性65 kDa蛋白（RPE65）在眼睛的视觉循环中起关键作用。Rpe65基因敲除（-/-）小鼠因缺乏11-顺式视黄醛、M-视蛋白降解和S-视蛋白定位错误而出现视力丧失。多项研究表明，9-顺式视黄醛和全反式视黄醛的前体9-顺式β-胡萝卜素可能在视力丧失的治疗中具有应用价值。因此，我们使用Rpe65（-/-）小鼠视网膜外植体培养物，研究了富含9-顺式β-胡萝卜素的巴氏杜氏藻是否能防止M-视蛋白的降解。
摘除三周龄Rpe65（-/-）和C57BL/6 J小鼠的眼睛，去除角膜。将眼杯在无或有巴氏杜氏藻的培养基中孵育6小时至4天。通过免疫组织化学观察视网膜中M-视蛋白的定位。通过蛋白质免疫印迹分析评估M-视蛋白、S-视蛋白和视紫红质蛋白的表达水平。
在C57BL/6 J小鼠视网膜中，外植体培养系统中M-视蛋白的定位和表达水平未观察到变化。在Rpe65（-/-）小鼠视网膜中，培养6小时至4天后，光感受器外段中M-视蛋白的量减少。然而，巴氏杜氏藻的存在显著改善了这种减少。相比之下，在外植体培养存在的情况下，S-视蛋白和视紫红质的表达水平没有变化。
这些结果表明，巴氏杜氏藻处理可防止Rpe65（-/-）小鼠视网膜中的M-视蛋白降解，并表明巴氏杜氏藻对由Rpe65基因突变引起的视网膜变性具有治疗潜力，如莱伯先天性黑蒙和色素性视网膜炎。
