Zhou Jie, Rappaport Eric F, Tobias John W, Young Terri L
Division of Ophthalmology, Children's Hospital of Philadelphia, Pennsylvania, USA.
Invest Ophthalmol Vis Sci. 2006 May;47(5):1794-802. doi: 10.1167/iovs.05-0759.
Ocular development involves changes in extracellular matrix components of the scleral wall as it expands. This study was conducted to determine scleral gene expression profiles during mouse ocular development to identify genes involved in normal scleral growth.
Sample sets of pooled sclerae of 3- and 8-week-old mice were microdissected, and total RNA was isolated. After reverse transcription, the cDNA was in vitro transcribed to produce biotin-labeled cRNA. The purified biotin-labeled cRNA samples were hybridized to microarray chips (GeneChip Mouse Genome 430 2.0; Affymetrix, Santa Clara, CA). Gene transcript expression profiles were determined, and eight differentially expressed genes between the two age groups underwent further confirmation by real-time PCR analysis.
Differential regulation of 4884 gene transcripts in mouse sclera with less than 5% false-discovery rate (FDR) was identified. The top 1000 with the lowest FDR among the 4884 probe sets were filtered for threefold changes between the two age groups, and 718 gene transcripts were identified. Among these 718 gene transcripts, 210 were upregulated and 508 downregulated in adult relative to juvenile mouse sclera. TGF-beta1 and several collagen genes were significantly downregulated. Microarray differential expression by real-time PCR validation of eight extracellular matrix-associated gene transcripts was confirmed.
This is the first study to demonstrate gene expression profiles in mouse sclera during ocular growth. These findings support the role of TGFbeta1 as a signaling molecule in modulating extracellular matrix during ocular development. This endeavor may be helpful in furthering understanding of how scleral remodeling is regulated during eye growth.
随着巩膜壁扩张，眼部发育涉及巩膜壁细胞外基质成分的变化。本研究旨在确定小鼠眼部发育过程中巩膜的基因表达谱，以识别参与正常巩膜生长的基因。
对3周龄和8周龄小鼠的巩膜样本进行显微切割，分离总RNA。逆转录后，将cDNA进行体外转录以产生生物素标记的cRNA。纯化后的生物素标记cRNA样本与微阵列芯片（GeneChip Mouse Genome 430 2.0；Affymetrix，加利福尼亚州圣克拉拉）杂交。确定基因转录本表达谱，两个年龄组之间差异表达的8个基因通过实时PCR分析进行进一步验证。
在小鼠巩膜中鉴定出4884个基因转录本的差异调节，错误发现率（FDR）小于5%。在4884个探针组中，FDR最低的前1000个被筛选出两个年龄组之间的三倍变化，鉴定出718个基因转录本。在这718个基因转录本中，相对于幼年小鼠巩膜，成年小鼠巩膜中有210个上调，508个下调。TGF-β1和几个胶原蛋白基因显著下调。通过实时PCR验证八个细胞外基质相关基因转录本的微阵列差异表达得到证实。
这是第一项证明小鼠眼部生长过程中巩膜基因表达谱的研究。这些发现支持TGFβ1作为信号分子在眼部发育过程中调节细胞外基质的作用。这项工作可能有助于进一步了解眼部生长过程中巩膜重塑是如何被调节的。
