Identification of genes involved in emerging cellular properties of corneal epithelial stem cells and its specific differentiation

为了鉴定在角膜上皮干细胞中赋予特殊细胞特性的基因，我们在角膜、角膜缘和结膜3层上皮细胞中进行了基因表达分析。为了选择性收集角膜缘基底上皮细胞，这被认为是假定的干细胞的角膜上皮细胞，我们使用激光显微切割装置。对于这样的分析，主要的障碍是有限的样品量;估计的最大RNA产量可能高达1 ng总RNA。因此，我们优化了iAFLP方法，从激光切割的冷冻组织片段中提取的微量RNA中精确测定基因表达谱。经过多次试验和错误实验，我们证实了优化的iAFLP方法可以调整到这样的小RNA。对9个样本中约500个角膜上皮特异性基因的基因表达分析允许鉴定在角膜缘基底上皮细胞中特异性表达的几个基因。这些基因包括整合素α 6、α 7、钙激活氯离子通道2（CLCA 2）、NGF受体（p75 NTR）、BAD、TNFR 2、THBS 3和角蛋白18。其中，p75 NTR在角膜和利姆布斯中的表达，显示出非常有趣的组织分布。该基因在角膜缘基底上皮细胞中呈显性特异性表达，提示该基因可能作为角膜上皮干细胞的特异性标志物。此外，它的对应基因，TrkA，表现出非常一致的分布p75 NTR，强烈暗示NOF，神经营养生长因子和这些二聚体受体的配体在维持和呈现干细胞特性的潜在功能的可能性。

To identify genes to render special cellular properties in stem cells of corneal epithelium, we performed gene expression analysis among 3 layers of corneal, limbal and conjunctival epithelial cells. For selective collection of limbal basal epithelial cells which are thought to be putative stem cells of corneal epithelial cells, we used a laser micro-dissection device. For such analysis, the major obstacles are limited amount of sample ; the estimated maximum yield of RNA might be as much as 1ng of total RNA. Thus we optimized iAFLP method for precise determination of gene expression profile from tiny amounts of RNA, which were extracted from laser dissected frozen tissues fragments. After several trial and error experiments, we confirmed the optimized iAFLP method to be adjusted to such small RNA. Gene expression analysis among the 9 samples against approx 500 corneal epithelium-specific genes allowed the identification of several genes which were specifically expressed in limbal basal epithelial cells. These genes included integrin alpha6,alpha7,calcium activated chloride channel 2 (CLCA2), NGF receptor (p75NTR), BAD, TNFR2,THBS3 and keratin 18. Among them, p75 NTR exhibited a very intriguing tissue distribution when we observed its expression in cornea and limbus by immunohistochemistry. Tissue distribution of the gene showed dominant and specific expression in limbal basal epithelial cells, suggesting the possibility of this gene as a specific marker for the corneal epithelial stem cells. Furthermore, its counterpart gene, TrkA, exhibited a very consistent distribution to the p75NTR, strongly implying the possibility of the potential function of NOF, a neurotrophic growth factor and a ligand for these dimeric receptors in maintaining and rendering the stem cell property.