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表现特殊细胞特性的基因，我们对角膜、角膜缘和结膜上皮细胞的3层细胞进行了基因表达分析。为了选择性地收集被认为是角膜上皮细胞干细胞的角膜缘基底上皮细胞，我们使用了激光显微解剖装置。对于这种分析，主要障碍是样本数量有限；估计RNA的最大产量可能高达总RNA的1ng。因此，我们优化了iAFLP方法，以精确测定从激光解剖冷冻组织碎片中提取的微量RNA的基因表达谱。经过多次试错实验，我们证实优化后的iAFLP方法可以适应如此小的RNA。通过对9个样本中约500个角膜上皮特异性基因的基因表达分析，鉴定出了几个在角膜缘基底上皮细胞中特异性表达的基因。这些基因包括整合素alpha6、alpha7、钙活化氯离子通道2 （CLCA2）、NGF受体（p75NTR）、BAD、TNFR2、THBS3和角蛋白18。其中p75 NTR通过免疫组化观察其在角膜和角膜缘的表达，呈现出非常有趣的组织分布。该基因的组织分布在角膜缘基底上皮细胞中显示显性和特异性表达，提示该基因可能作为角膜上皮干细胞的特异性标记物。此外，其对应基因TrkA与p75NTR的分布非常一致，这强烈暗示了NOF作为一种神经营养生长因子和二聚体受体的配体在维持和呈现干细胞特性方面的潜在功能。

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.