Institute for Ophthalmic Research, Section of Neurobiology of the Eye, University of Tübingen, Tübingen, Germany.
Invest Ophthalmol Vis Sci. 2010 Jul;51(7):3726-35. doi: 10.1167/iovs.09-4615. Epub 2010 Mar 5.
PURPOSE. Ocular growth is regulated locally by signals produced in the retina. The highly heterogeneous nature of the retina may mask important changes in gene expression during global analysis. This study was conducted to investigate changes in gene expression specifically within the amacrine cell layer (ACL), the most likely generator of growth signals, during optical manipulation of ocular growth. METHOD. Chicks were monocularly treated with either -7-D (n = 6) or +7-D (n = 6) lenses for 24 hours. Untreated age-matched chicks served as control subjects (n = 6). Total RNA from the ACL was isolated from 10-mum-thick sections, obtained using laser capture microdissection. Labeled cRNA was prepared from three samples per condition and hybridized to chicken genome microarrays. Changes in gene expression were validated by using semiquantitative real-time RT-PCR. RESULTS. One hundred twenty-eight genes were differentially expressed in the ACL of the minus lens-treated eyes, whereas the plus lens-treated eyes displayed 58 changes 24 hours after treatment. Only 11 genes were differentially expressed under both experimental conditions, whereas the expression of only one gene (clone ChEST927g14) was modulated by the sign of defocus. Compared with previous studies in the field, the magnitude of changes observed in the present work were larger, with more than 30% of differentially expressed genes showing a twofold or greater modulation in expression. The results, obtained from independent validation by real-time RT-PCR technology, correlated highly with the original microarray data. The differential expression of four of eight genes was validated in plus lens-treated eyes, and eight of nine genes were independently validated in minus lens-treated eyes. CONCLUSIONS. The targeted investigation of the ACL enabled the identification of several novel genes that may form part of the growth regulatory pathways of the eye. Different retinal pathways may underlie the response of the eyes to plus and minus lens compensation, as there was limited overlap in the regulated genes observed within the ACL under both conditions.
眼球的生长受到视网膜中产生的信号的局部调控。由于视网膜具有高度异质性，因此在进行全局分析时可能会掩盖基因表达的重要变化。本研究旨在探讨在光学操纵眼球生长过程中，特定于无长突细胞层（amacrine cell layer，ACL）的基因表达变化，因为 ACL 最有可能产生生长信号。
鸡的单眼分别用-7-D（n = 6）或+7-D（n = 6）透镜处理 24 小时。未处理的同龄小鸡作为对照（n = 6）。使用激光捕获显微切割术从 10 微米厚的切片中分离 ACL 的总 RNA。每个条件下制备三个样本的标记 cRNA 并杂交到鸡基因组微阵列上。使用半定量实时 RT-PCR 验证基因表达的变化。
在负透镜处理眼的 ACL 中，有 128 个基因差异表达，而在正透镜处理眼的 ACL 中，有 58 个基因在处理后 24 小时发生变化。只有 11 个基因在两种实验条件下差异表达，而只有一个基因（clone ChEST927g14）的表达受离焦的符号调节。与该领域以前的研究相比，本研究观察到的变化幅度更大，超过 30%的差异表达基因的表达被调节两倍或更多。通过实时 RT-PCR 技术进行的独立验证的结果与原始微阵列数据高度相关。在正透镜处理眼中验证了 8 个基因中的 4 个，在负透镜处理眼中独立验证了 9 个基因中的 8 个。
对 ACL 的靶向研究鉴定了几个可能成为眼部生长调节途径一部分的新基因。不同的视网膜途径可能是眼睛对正、负透镜补偿反应的基础，因为在两种条件下，ACL 中观察到的受调控基因之间的重叠有限。
