Pfeffer Bruce A, Becerra S Patricia, Borst Diane E, Wong Paul
Research, Bausch and Lomb, Inc., Rochester, NY 14609, USA.
Mol Vis. 2004 Jan 14;10:23-30.
To document the expression of mRNA for transthyretin (TTR) and retinol binding protein (RBP) in native and cultured Rhesus monkey retinal pigmented epithelium (RPE); to compare mRNA transcripts for these two proteins expressed in RPE with those found in whole monkey liver and brain; to demonstrate the secretion of TTR by RPE during short-term maintenance in a protein-free, defined medium, as a manifestation of the differentiated state of these cells in vitro.
Total RNA was isolated from cultured RPE in first passage, after incubation for eight days in defined, protein-free medium. Conditioned medium was collected for western analysis at this time. Total RNA was also extracted from RPE/choroid freshly dissected from monkey eyes. Using cDNA probes for human TTR and RBP, northern analysis was performed on the total RNA from fresh and cultured RPE samples, together with poly(A+) mRNA purified from monkey liver and brain.
Conditioned medium from RPE yielded TTR protein of the expected monomer subunit molecular size. The TTR secreted de novo from the cultured cells was detectable in the absence of biosynthetic labeling. With the exception of some extremely low abundance transcripts expressed in cultured RPE, all samples contained a single 900 bp transcript for TTR. Based on relative amounts of actual message, RPE ranks higher than liver in abundance of TTR mRNA. In contrast, both native monkey RPE and cultured RPE cells expressed comparatively low levels of mRNA for RBP. All samples displayed a single RBP mRNA transcript at 1100 bp.
Our results indicate that TTR is a significant gene product of the RPE, and may be considered as a marker for a differentiated phenotype for these cells in culture. There is increased recognition of various forms of ocular pathology associated with mutations or other malfunctions involving TTR and RBP, warranting a greater understanding of mechanisms of transcriptional and translational control for these two proteins.
记录运甲状腺素蛋白（TTR）和视黄醇结合蛋白（RBP）的mRNA在原代和培养的恒河猴视网膜色素上皮（RPE）中的表达；比较RPE中表达的这两种蛋白质的mRNA转录本与在整个猴肝脏和大脑中发现的转录本；证明RPE在无蛋白限定培养基中短期培养期间分泌TTR，作为这些细胞在体外分化状态的一种表现。
从第一代培养的RPE中分离总RNA，这些细胞在限定的无蛋白培养基中孵育八天后。此时收集条件培养基用于蛋白质印迹分析。还从猴眼中新鲜分离的RPE/脉络膜中提取总RNA。使用针对人TTR和RBP的cDNA探针，对新鲜和培养的RPE样品的总RNA以及从猴肝脏和大脑中纯化的聚腺苷酸（A+）mRNA进行Northern印迹分析。
RPE的条件培养基产生了预期单体亚基分子大小的TTR蛋白。在没有生物合成标记的情况下，可以检测到从培养细胞中新分泌的TTR。除了在培养的RPE中表达的一些极低丰度转录本外，所有样品都含有一个900bp的TTR转录本。基于实际信息的相对量，RPE中TTR mRNA的丰度高于肝脏。相比之下，原代猴RPE和培养的RPE细胞中RBP的mRNA表达水平相对较低。所有样品在1100bp处显示单一的RBP mRNA转录本。
我们的结果表明，TTR是RPE的一种重要基因产物，可被视为这些培养细胞分化表型的标志物。人们越来越认识到与涉及TTR和RBP的突变或其他功能障碍相关的各种形式的眼部病理学，这需要更深入地了解这两种蛋白质的转录和翻译控制机制。
