Sundelin S, Wihlmark U, Nilsson S E, Brunk U T
Department of Ophthalmology, Linköping University, Sweden.
Curr Eye Res. 1998 Aug;17(8):851-7.
Retinal pigment epithelial (RPE) cells slowly accumulate lipofuscin pigment within their acidic vacuolar apparatus as a result of extra- and/or intralysosomal oxidative alterations of phagocytosed photoreceptor outer segments (POS) with consequent imperfect degradation of these structures. In old age, lipofuscin accumulation may become quite substantial. It has been suggested that pronounced accumulation of lipofuscin is related to decreased RPE function and, possibly, to age-related macular degeneration. The aim of the present investigation was to study whether heavy loading with lipofuscin of RPE acidic lysosomes would affect the further phagocytic ability of the cells.
In the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS (artificial lipofuscin) for 4 weeks, resulting in a pronounced lipofuscin accumulation of the cells. Fluorescent latex beads (labelled with a red fluorophore) were added to unloaded control cultures at O and 4 weeks after their establishment, and to lipofuscin loaded cells after 4 weeks of feeding with artificial lipofuscin. Cellular amounts of lipofuscin, and their phagocytotic activity, were quantified by static fluorometry measuring lipofuscin-specific and red bead-specific fluorescence, respectively. The intracellular location of the beads was verified by confocal laser scanning microscopy.
Unloaded, and thus almost lipofuscin-free, control cells exposed to latex beads showed numerous cytoplasmic particles emitting reddish fluorescence, while few particles were taken up by cells initially loaded with artificial, POS-derived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significantly (p < 0.001) higher levels in unloaded control cells than in lipofuscin-loaded ones. In the second part of the investigation, unloaded control cultures and lipofuscin-loaded cultures were exposed to native bovine Texas Red-X-labelled POS 4 weeks after the establishment of the cultures. Unloaded control cells showed a large number of cytoplasmic POS emitting reddish fluorescence, while fewer POS were phagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red-X-specific fluorescence, thus quantifying the phagocytic ability of the cells, showed significantly (p < 0.001) higher levels in control cells than in lipofuscin-loaded ones.
Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity. The resulting reduction in ability to cope with the needs of the overlying photoreceptor cells, in order to eliminate the obsolete tips of their POS, may well be of significance in the development of age-related macular degeneration.
视网膜色素上皮（RPE）细胞由于吞噬的光感受器外段（POS）在溶酶体外和/或溶酶体内发生氧化改变，导致这些结构降解不完全，从而在其酸性液泡装置中缓慢积累脂褐素色素。在老年时，脂褐素的积累可能会相当显著。有人提出，脂褐素的大量积累与RPE功能下降以及可能与年龄相关性黄斑变性有关。本研究的目的是探讨RPE酸性溶酶体中大量负载脂褐素是否会影响细胞的进一步吞噬能力。
在研究的第一部分，将兔RPE细胞培养物每天暴露于经紫外线照射的牛POS（人工脂褐素）中4周，导致细胞中脂褐素显著积累。在未负载的对照培养物建立后的第0周和第4周，以及在用人工脂褐素喂养4周后的脂褐素负载细胞中加入荧光乳胶珠（用红色荧光团标记）。通过静态荧光测定法分别测量脂褐素特异性荧光和红色珠特异性荧光，对脂褐素的细胞含量及其吞噬活性进行定量。通过共聚焦激光扫描显微镜验证珠子在细胞内的位置。
暴露于乳胶珠的未负载且几乎不含脂褐素的对照细胞显示出许多发出微红荧光的细胞质颗粒，而最初负载有人工POS来源的脂褐素的细胞摄取的颗粒很少。乳胶珠特异性荧光的测量显示，未负载的对照细胞中的水平显著（p < 0.001）高于脂褐素负载细胞。在研究的第二部分，在培养物建立4周后，将未负载的对照培养物和脂褐素负载培养物暴露于天然牛德克萨斯红-X标记的POS。未负载的对照细胞显示出大量发出微红荧光的细胞质POS，而负载人工脂褐素的细胞吞噬的POS较少。对德克萨斯红-X特异性荧光的测量，从而量化细胞的吞噬能力，显示对照细胞中的水平显著（p < 0.001）高于脂褐素负载细胞。
RPE细胞中严重的脂褐素积累似乎导致吞噬能力大大降低。为了消除其POS的过时末端，应对上层光感受器细胞需求的能力下降很可能在年龄相关性黄斑变性的发展中具有重要意义。
