Rabin David M, Rabin Richard L, Blenkinsop Timothy A, Temple Sally, Stern Jeffrey H
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany NY 12208, USA.
Aging (Albany NY). 2013 Jan;5(1):51-66. doi: 10.18632/aging.100516.
The goal of this study was to examine changes in the expression of transcripts and proteins associated with drusen in Age-related Macular Degeneration (AMD) after exposing human retinal pigment epithelium (hRPE) cells to chronic oxidative stress.
Primary adult human RPE cells were isolated from cadaveric donor eyes. The subpopulation of RPE stem cells (RPESCs) was activated, expanded, and then differentiated into RPE progeny. Confluent cultures of RPESC-derived hRPE and ARPE-19 cells were exposed to a regimen of tert-butylhydroperoxide (TBHP) for 1-5 days. After treatment, gene expression was measured by quantitative PCR (qPCR), protein expression was assessed by immunocytochemistry and transepithelial resistance and cell toxicity were measured.
hRPE cells exposed to a regimen of TBHP for 5 days upregulate expression of several molecules identified in drusen, including molecular chaperones and pro-angiogenic factors. 5-day TBHP treatment was significantly more effective than 1-day treatment at eliciting these effects. The extent of hRPE response to 5-day treatment varied significantly between individual donors, nevertheless, 6 transcripts were reliably significantly upregulated. ARPE-19 cells treated with the same 5-day stress regime did not show the same pattern of response and did not upregulate this group of transcripts.
RPESC-derived hRPE cells change significantly when exposed to repeated oxidative stress conditions, upregulating expression of several drusen-related proteins and transcripts. This is consistent with the hypothesis that hRPE cells are competent to be a source of proteins found in drusen deposits. Our results suggest that donor-specific genetic and environmental factors influence the RPE stress response. ARPE-19 cells appear to be less representative of AMD-like changes than RPESC-derived hRPE. This adult stem cell-based system using chronic TBHP treatment of hRPE represents a novel in vitro model useful for the study of drusen formation and dry AMD pathophysiology.
本研究的目的是检测在将人视网膜色素上皮（hRPE）细胞暴露于慢性氧化应激后，年龄相关性黄斑变性（AMD）中与玻璃膜疣相关的转录本和蛋白质表达的变化。
从尸体供体眼中分离出原代成人RPE细胞。激活、扩增RPE干细胞（RPESC）亚群，然后将其分化为RPE子代。将RPESC来源的hRPE和ARPE - 19细胞的汇合培养物暴露于叔丁基过氧化氢（TBHP）方案1 - 5天。处理后，通过定量PCR（qPCR）测量基因表达，通过免疫细胞化学评估蛋白质表达，并测量跨上皮电阻和细胞毒性。
暴露于TBHP方案5天的hRPE细胞上调了在玻璃膜疣中鉴定出的几种分子的表达，包括分子伴侣和促血管生成因子。5天的TBHP处理在引发这些效应方面比1天的处理显著更有效。hRPE对5天处理的反应程度在个体供体之间有显著差异，然而，有6种转录本确实可靠地显著上调。用相同的5天应激方案处理的ARPE - 19细胞没有显示出相同的反应模式，并且没有上调这组转录本。
当暴露于反复的氧化应激条件时，RPESC来源的hRPE细胞发生显著变化，上调了几种与玻璃膜疣相关的蛋白质和转录本的表达。这与hRPE细胞能够成为玻璃膜疣沉积物中发现的蛋白质来源的假设一致。我们的结果表明，供体特异性的遗传和环境因素会影响RPE应激反应。ARPE - 19细胞似乎比RPESC来源的hRPE更不能代表AMD样变化。这种使用慢性TBHP处理hRPE的基于成体干细胞的系统代表了一种用于研究玻璃膜疣形成和干性AMD病理生理学的新型体外模型。
