mRNA expression by RPE cells in myopia

描述(申请人提供)：许多证据表明，视网膜在很大程度上调节眼睛的生长和屈光不正的发展，但对视网膜在体内控制巩膜生长的细胞通路和信号级联的分子成分的识别还知之甚少。我们建议研究单个视网膜色素上皮(RPE)细胞的mRNA表达，以解决局部视网膜到巩膜信号在眼睛生长控制中的作用，因为RPE细胞在感觉性视网膜和脉络膜/巩膜之间形成中间屏障层，并且根据现有数据，可以局部介导视网膜对屈光发育的影响。我们将通过应用微阵列技术对从不同眼底区域选择的单个RPE细胞的基因表达进行研究，这些细胞是从两个成熟的鸡眼生长模型中挑选出来的。 结合一个跨学科的研究团队和宾夕法尼亚大学的核心设施，我们提出了以下相互关联的具体目标：1)从位于眼睛生长模型不同眼底区域的单个RPE细胞的mRNA中制备ARNA；2)完成并利用高密度鸡RPE/视网膜微阵列从RPE细胞中提取ArNA；3)采用信息学方法分析单个RPE细胞的基因表达；以及4)验证细胞基因表达谱。 通过根据RPE细胞的位置确定mRNA的表达模式，我们预计结果将进一步涉及RPE细胞的生长调节过程，并提供关于屈光发育中视网膜到巩膜的局部信号的新的分子和空间信息。此外，证明单细胞信使核糖核酸图谱用于屈光研究的可行性和实用性可以极大地促进现有技术的发展，并为该领域未来的细胞和分子研究提供基础。我们相信，这项计划将有助于更好地了解屈光不正的病因，并最终找到有效的方法来遏制儿童近视。

DESCRIPTION (provided by applicant): Much evidence indicates that the retina largely regulates eye growth and the development of refractive errors, but the identity of both the cellular pathways and the molecular components of the signaling cascade by which the retina controls scleral growth in vivo are poorly understood. We propose to investigate mRNA expression by individual retinal pigment epithelial (RPE) cells to address local retina-to-sclera signaling in eye growth control because RPE cells form the intervening barrier layer between the sensory retina and the choroid/sclera and, based on available data, could locally mediate the retinal influences on refractive development. We shall study gene expression in individual RPE cells selected from different fundus regions in two well-established eye growth models in the chick by applying microarray profiling to amplified antisense-RNA (aRNA) prepared from the mRNA of these cells. Combining an interdisciplinary research team and University of Pennsylvania core facilities, we propose the following inter-related Specific Aims: 1) prepare aRNA from the mRNA of individual RPE cells located at different fundus regions in eye growth models; 2) complete and then utilize high density chick RPE/retinal microarrays to profile aRNA from RPE cells; 3) adapt informatics approaches for analysis of gene expression by individual RPE cells; and 4) validate the cellular gene expression profiles. By identifying mRNA expression patterns according to RPE cell location, we anticipate that the results will further implicate RPE cells in the growth regulatory process and provide novel molecular and spatial information about the local signaling from retina to sclera in refractive development. Further, demonstrating the feasibility and utility of single cell mRNA profiling for refractive research could substantially advance the available techniques and provide the foundation for future cell and molecular studies in the area. We believe this program will lead to improved understanding of the etiology of refractive errors and ultimately to effective approaches to arrest myopia in children.