Corneal Dystrophy Research Institute, Department of Ophthalmology, College of Medicine, Yonsei University, Seoul, Korea.
Invest Ophthalmol Vis Sci. 2010 Apr;51(4):1832-47. doi: 10.1167/iovs.09-4149. Epub 2009 Nov 20.
Purpose. The purpose of this study was to elucidate the pathophysiological process in primary cultured corneal fibroblasts (PCFs) from normal subjects and granular corneal dystrophy (GCD) II patients, by using cDNA microarrays. Methods. PCFs were isolated from the corneas of normal subjects and GCD II patients who were heterozygous and homozygous for the TGFBI R124H mutation. RNA was isolated from each sample, and gene expression profiles were analyzed with a cDNA microarray consisting of approximately 29,000 genes. Cell adhesion assays were performed to confirm the functionality of the detected gene expression profiles. Results. Twofold differences were detected in the expression of 555 genes between wild-type and homozygous GCD II PCFs. Of these, 319 genes were upregulated, and 236 genes were downregulated in the homozygous GCD II PCFs. The most abundant and consistent changes were observed in gene families encoding signal transduction pathways involving the TGF-beta receptor- and integrin-mediated signaling, cell differentiation and proliferation, immune responses, cell adhesion, extracellular matrix (ECM) proteolytic enzymes, cell cycle, cytoskeletal organization, mitochondrial energy metabolism, collagen catabolism, response to wounding, response to oxidative stress, and the ubiquitin-mediated proteasomal degradation pathway. Cell adhesion assays demonstrated that heterozygous and homozygous GCD II PCFs strongly attached to collagen-I, collagen-IV, fibronectin, and laminin, compared with wild-type cells. Conclusions. Alterations in the TGF-beta receptor- and integrin-mediated signaling pathway may play a key role in GCD II pathophysiology. If the novel factors identified in this study are involved in GCD II pathogenesis, they could assist in designing further studies to elucidate specific mechanisms of this disease.
目的。本研究旨在通过 cDNA 微阵列阐明正常受试者和颗粒状角膜营养不良（GCD）II 患者原代培养角膜成纤维细胞（PCF）的病理生理过程。方法。从正常受试者和 GCD II 患者的角膜中分离出 PCF，这些患者分别为 TGFBI R124H 突变的杂合子和纯合子。从每个样本中分离 RNA，并使用包含约 29000 个基因的 cDNA 微阵列分析基因表达谱。进行细胞黏附实验以验证所检测的基因表达谱的功能。结果。在野生型和纯合 GCD II PCF 之间，检测到 555 个基因的表达存在两倍差异。其中，319 个基因在纯合 GCD II PCF 中上调，236 个基因下调。在涉及 TGF-β受体和整合素介导的信号转导、细胞分化和增殖、免疫反应、细胞黏附、细胞外基质（ECM）蛋白水解酶、细胞周期、细胞骨架组织、线粒体能量代谢、胶原分解代谢、对创伤的反应、对氧化应激的反应以及泛素介导的蛋白酶体降解途径的基因家族中观察到最丰富和一致的变化。细胞黏附实验表明，与野生型细胞相比，杂合子和纯合子 GCD II PCF 强烈黏附于胶原-I、胶原-IV、纤连蛋白和层粘连蛋白。结论。TGF-β受体和整合素介导的信号通路的改变可能在 GCD II 病理生理学中起关键作用。如果本研究中鉴定的新因子参与 GCD II 的发病机制，它们可能有助于设计进一步的研究以阐明该疾病的特定机制。
