A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture.
A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture.
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DOI：
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发表时间：
1999-11
影响因子：
4.4
通讯作者：
J. Sparrow;C. Parish;M. Hashimoto;Koji Nakanishi
中科院分区：
文献类型：
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作者：
J. Sparrow;C. Parish;M. Hashimoto;Koji Nakanishi
目的研究视网膜色素上皮（RPE）细胞脂褐素（lipofuscin）的一种成分A2 E被培养的人RPE细胞内化后的变化。方法合成A2 E并与缺乏天然脂褐素的成人RPE细胞系孵育。为了研究A2 E的细胞区室化，将细胞与A2 E和荧光亲酸探针（Lysotracker Red DND-99; Molecular Probes，尤金，OR）同时孵育。通过测定细胞质酶乳酸脱氢酶（LDH）的泄漏，用膜不渗透染料进行荧光核染色和形态学标准来评价质膜完整性。还测定了内化A2 E的发射光谱。通过高效液相色谱法定量由培养细胞积累的A2 E水平，并与从人眼分离的RPE中存在的量进行比较。结果A2 E被RPE细胞内化，通过荧光共聚焦成像可检测到细胞内颗粒的获得。内化的A2 E具有565至570 nm的发射最大值。与10至25 μ M A2 E孵育的细胞中积累的A2 E水平与从人眼收获的等量RPE细胞中存在的A2 E量相当。A2 E和Lysotracker探针的共定位揭示了在酸性细胞器中的优先积累。暴露于50和100 μ M A2 E后测得的LDH水平升高可归因于A2 E蓄积细胞亚群中的膜损伤，通过荧光核标记测定。结论：内化A2 E对酸性细胞器具有亲和力。由A2 E积累RPE表现出的膜损伤取决于A2 E的浓度，并反映了这种两亲性化合物发挥洗涤剂样作用的能力。
PURPOSE To study A2E, a component of retinal pigmented epithelial (RPE) cell lipofuscin, after its internalization by cultured human RPE cells. METHODS A2E was synthesized and incubated with an adult RPE cell line devoid of native lipofuscin. To investigate the cellular compartmentalization of A2E, cells were incubated simultaneously with A2E and a fluorescent acidotropic probe, (Lysotracker Red DND-99; Molecular Probes, Eugene, OR). Plasma membrane integrity was evaluated by assaying for leakage of the cytoplasmic enzyme lactate dehydrogenase (LDH), by fluorescence nuclear staining with a membrane-impermeant dye and by morphologic criteria. The emission spectrum of internalized A2E was also determined. The levels of A2E accumulated by the cultured cells were quantified by high-performance liquid chromatography and compared with amounts present in RPE isolated from human eyes. RESULTS Internalization of A2E by the RPE cells was evidenced by the acquisition of intracellular granules detectable by fluorescence confocal imaging. Internalized A2E had an emission maxima of 565 to 570 nm. The levels of A2E accumulating in cells incubated with 10 to 25 microM A2E were comparable to the amounts of A2E present in equal numbers of RPE cells harvested from human eyes. Colocalization of A2E and the Lysotracker probe revealed a preferential accumulation in acidic organelles. The elevated LDH levels that were measured after exposure to 50 and 100 microM A2E were attributable to membrane damage in a subpopulation of the A2E-accumulating cells, determined by fluorescence nuclear labeling. CONCLUSIONS Internalized A2E has an affinity for acidic organelles. The membrane damage exhibited by A2E-accumulating RPE is dependent on the concentration of A2E and reflects the ability of this amphiphilic compound to exert detergent-like effects.