Gene Expression Profile of Extracellular Matrix and Adhesion Molecules in the Human Normal Corneal Stroma

Purpose: There is limited information on region-specific gene expression in the human corneal stroma. In this study, we aimed to investigate the expression profile of the extracellular matrix and adhesion molecules in the normal corneal stroma using laser capture microdissection (LCM) and molecular techniques.
Methods: Frozen sections of human cornea without ocular disease were used to isolate the central and peripheral corneal stromal keratocytes by LCM. RNA was extracted from LCM-captured tissues and the RT2 Profiler PCR Arrays were used to examine the expression profile of extracellular matrix and adhesion molecules in the central and peripheral stroma. Real-time quantitative PCR was used to quantify gene expression. Proteomic and western blotting (WB) analyses were performed to confirm gene expression at protein level. Function association network was generated via the web tools String and Cytoscape.
Results: The gene expression profiling demonstrated that 35 out of the 84 extracellular matrix and adhesion molecules represented in the array were expressed in stromal keratocytes. Among them, 24 genes were not previously described in the corneal stroma. Two genes were found more abundantly expressed in the central stroma than in the periphery: TGFBI, COL6A2 (p < 0.05). ADAMTS13 was detected only in the central stroma. Proteomics and WB analysis confirmed the expression of 10 genes. Functional analysis revealed that most identified genes were presented in a core cluster that had multiple and strong associations with other genes.
Conclusion: This study identified genes not previously described in the corneal stroma, revealed regional differences in gene expression between central and peripheral stroma, and also detected some interesting candidate genes that may play important roles in corneal function. These observations serve as the foundation to further investigate the molecular and cellular mechanisms regulating the pathogenesis of regional corneal stromal disorders such as keratoconus.

研究目的：目前关于人类角膜基质区域特异性基因表达的相关信息较为匮乏。本研究旨在利用激光捕获显微切割（Laser Capture Microdissection, LCM）技术与分子生物学手段，探究正常角膜基质中细胞外基质（extracellular matrix）与黏附分子（adhesion molecules）的表达谱。
研究方法：选取无眼部疾病的人角膜冰冻切片，通过LCM技术分离中央与周边角膜基质角膜细胞。从LCM捕获的组织中提取RNA，采用RT2 Profiler PCR阵列检测中央与周边角膜基质中细胞外基质及黏附分子的表达谱。通过实时定量PCR（Real-time Quantitative PCR）对基因表达进行定量分析，并开展蛋白质组学与蛋白质印迹（Western Blotting, WB）分析以在蛋白层面验证基因表达情况。此外，通过String与Cytoscape在线工具构建功能关联网络。
研究结果：基因表达谱分析显示，该阵列所涵盖的84种细胞外基质与黏附分子中，有35种在角膜基质细胞中表达。其中24种基因此前未见角膜基质相关报道。相较于周边基质，有2种基因在中央基质中表达量更高：TGFBI、COL6A2（p < 0.05）。ADAMTS13仅在中央基质中被检测到。蛋白质组学与WB分析验证了其中10种基因的表达。功能关联分析显示，多数已鉴定基因聚集于一个核心基因簇，该基因簇与其他基因存在多且强的相互关联。
研究结论：本研究鉴定出此前未在角膜基质中报道过的基因，揭示了中央与周边角膜基质间的基因表达区域差异，同时发现了若干可能在角膜功能中发挥重要作用的潜在候选基因。上述研究结果可为进一步探究圆锥角膜（keratoconus）等区域性角膜基质疾病的发病相关分子与细胞机制奠定基础。