Rakoczy P E, Baines M, Kennedy C J, Constable I J
Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands.
Exp Eye Res. 1996 Aug;63(2):159-67. doi: 10.1006/exer.1996.0104.
The purpose of the present study was to investigate the accumulation of rod outer segment (ROS)-derived debris in cultured human retinal pigment epithelial cells (RPE). The RPE cell layer is responsible for the phagocytosis and digestion of photoreceptor outer segments. Due to the immense volume of photoreceptor-derived material processed by the RPE cells, even minor changes in the efficiency of ROS processing may cause the accumulation of lipofuscin and photoreceptor derived debris. In this work, 17 RPE cultures were established from the globes of eye bank donors whose ages ranged from 18 to 79 years. Third passage cultures were challenged with bovine ROS and the accumulation of an autofluorescent debris was quantified using a flow cytometer. It was demonstrated that ROS challenge greatly increased the rate of autofluorescent debris accumulation. The accumulation of autofluorescent debris varied significantly from culture to culture. This variation was independent of the phagocytosing capacity of individual cultures and was not age dependent. To further investigate the factors which may be responsible for these differences, the presence of cathepsin D, an aspartic protease responsible for 80% of proteolysis of rhodopsin, was analysed by Western blot. Although the 34 kDa active form of cathepsin D was found in all cultures, in 41% of the cultures higher-molecular-weight forms of cathepsin D were additionally present, thus providing a multimer form of cathepsin D in these cultures. The rate of autofluorescent debris accumulation in cultures possessing a multimer form of cathepsin D was significantly greater (mean 42.3, S.D. +/- 19.8) than those in cultures having a singlet active form (mean 18.8, S.D. +/- 5.5) at 34 kDa (Student's t-test, DF = 15, t = 6.834, P < 0.001). The former cultures included one from a donor with age related macular degeneration, the latter cultures included one from a donor with diabetic retinopathy. This study demonstrates that the rate of autofluorescent debris accumulation in cultured RPE cells is not age dependent, but is an intrinsic property of the donor RPE cells that is possibly related to the presence of a multimer form of the lysosomal enzyme cathepsin D.
本研究的目的是调查培养的人视网膜色素上皮细胞（RPE）中视杆细胞外段（ROS）衍生碎片的积累情况。RPE细胞层负责吞噬和消化光感受器外段。由于RPE细胞处理的光感受器衍生物质数量巨大，即使ROS处理效率的微小变化也可能导致脂褐素和光感受器衍生碎片的积累。在这项工作中，从年龄在18至79岁的眼库供体的眼球中建立了17个RPE培养物。第三代培养物用牛ROS进行刺激，并使用流式细胞仪对自发荧光碎片的积累进行定量。结果表明，ROS刺激大大增加了自发荧光碎片的积累速率。自发荧光碎片的积累在不同培养物之间有显著差异。这种差异与各个培养物的吞噬能力无关，也与年龄无关。为了进一步研究可能导致这些差异的因素，通过蛋白质印迹法分析了组织蛋白酶D的存在情况，组织蛋白酶D是一种天冬氨酸蛋白酶，负责80%的视紫红质蛋白水解。虽然在所有培养物中都发现了34 kDa的活性形式的组织蛋白酶D，但在41%的培养物中还额外存在高分子量形式的组织蛋白酶D，从而在这些培养物中提供了组织蛋白酶D的多聚体形式。具有组织蛋白酶D多聚体形式的培养物中自发荧光碎片的积累速率（平均值42.3，标准差±19.8）显著高于具有34 kDa单活性形式的培养物（平均值18.8，标准差±5.5）（学生t检验，自由度 = 15，t = 6.834，P < 0.001）。前一种培养物包括来自一名患有年龄相关性黄斑变性供体的培养物，后一种培养物包括来自一名患有糖尿病性视网膜病变供体的培养物。本研究表明，培养的RPE细胞中自发荧光碎片的积累速率与年龄无关，而是供体RPE细胞的一种内在特性，可能与溶酶体酶组织蛋白酶D的多聚体形式的存在有关。
