RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo
RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo
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DOI：
10.1167/iovs.04-0074
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发表时间：
2004-08-01
影响因子：
4.4
通讯作者：
Rodriguez, IR
中科院分区：
文献类型：
--
作者：
Gordiyenko, N;Campos, M;Rodriguez, IR
目的.确定血浆低密度脂蛋白（LDL）是否可被RPE内化，以及哪些受体可能参与。次要目的是确定ARPE 19细胞是否可以用作研究RPE中胆固醇加工的模型。市售的人LDL用罗丹明或AlexaFluor 568标记。使用市售的LDL-R、CD 36和LOX-1抗体进行免疫荧光。使用落射荧光和共聚焦荧光显微镜对细胞和组织进行成像。免疫印迹分析和RT-PCR采用已发表的技术进行。荧光共聚焦显微镜观察视网膜内注射罗丹明标记的低密度脂蛋白（rhoLDL）24 h后，视网膜色素上皮细胞中rhoLDL的表达。rhoLDL存在于某些区域，而在其他区域则不存在。培养的ARPE 19细胞也被发现容易内化LDL和氧化LDL（oxLDL）。使用AlexaFluor 568标记的LDL，我们确定平均培养的RPE细胞可以在24小时内内化约12至16 pg的LDL和oxLDL。免疫印迹法很容易检测到的CD 36和LDL-R的存在下，在培养的RPE细胞，但不是LOX-1，而RT-PCR检测所有三种受体的mRNA。用AlexaFluor 568标记的LDL和AlexaFluor 488对受体进行免疫定位的双标记实验显示LDL-R与内化的LDL和CD 36与oxLDL颗粒共定位。血浆LDL容易通过脉络膜毛细血管进入RPE，但在整个视网膜中并不均匀。这可能表明有孔脉络膜内皮细胞连接的渗透性的某种形式的调节。ARPE 19细胞是研究LDL和oxLDL内化机制的良好体外模型。LDL可以用作载体以将疏水分子携带到RPE中。
Purpose. To determine whether plasma low-density lipoprotein (LDL) could be internalized by the RPE and which receptors may be involved. A secondary objective was to determine whether ARPE19 cells could be used as a model to investigate cholesterol processing in the RPE.Methods. Commercially available human LDL was labeled with rhodamine or AlexaFluor 568. Immunofluorescence was performed using commercially available antibodies to LDL-R, CD36, and LOX-1. Cells and tissues were imaged using epifluorescence and confocal fluorescence microscopy. Immunoblot analysis and RT-PCR were performed using published techniques.Results. Intravenously injected rhodamine-labeled LDL (rhoLDL) was detected in the rat RPE by fluorescence confocal microscopy 24 hours after injection. The rhoLDL was present in some areas and absent in others. Cultured ARPE19 cells were also found to internalize LDL and oxidized LDL (oxLDL) readily. Using AlexaFluor 568-labeled LDL we determined that the average cultured RPE cell could internalize approximately 12 to 16 pg of LDL and oxLDL in 24 hours. Immunoblots readily detected the presence of CD36 and LDL-R in the cultured RPE cells but not LOX-1, whereas RT-PCR detected mRNA for all three receptors. Dual-labeling experiments using AlexaFluor 568-labeled LDL and AlexaFluor 488 for the immunolocalization of the receptors showed colocalization of LDL-R with the internalized LDL and CD36 with oxLDL particles.Conclusions. Plasma LDL readily enters the RPE through the choriocapillaris but is not found homogenously throughout the retina. This may suggest some form of regulation to the permeability of the fenestrated choroidal endothelial cell junctions. ARPE19 cells are a good model for studying the internalization mechanisms of LDL and oxLDL in vitro. LDL may be used as a vector to carry hydrophobic molecules into the RPE.