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

为了确定在角膜上皮干细胞中呈现特殊细胞特性的基因，我们对角膜、角膜缘和结膜上皮三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切割设备。对于这种分析，主要的障碍是样本量有限；估计RNA的最高产量可能高达总RNA的1 ng。因此，我们优化了iAFLP方法，以准确地从激光切割的冰冻组织片段中提取微量的RNA来确定基因表达谱。经过多次试错实验，我们确定了优化后的iAFLP方法适用于如此小的RNA。根据大约500个角膜上皮细胞特异性基因，在9个样本中进行基因表达分析，可以识别出几个在角膜缘基底上皮细胞中特异表达的基因。这些基因包括整合素α6、α7、钙激活氯通道2(CLCA2)、神经生长因子受体(P75NTR)、BAD、TNFR2、THBS3和角蛋白18。该基因在角膜缘上皮细胞中的组织分布呈显性和特异性表达，提示该基因有可能作为角膜上皮干细胞的特异性标志物。此外，其对应基因TrkA与p75NTR的分布非常一致，这强烈暗示了NOF的潜在功能，NOF是一种神经营养生长因子，也是这些二聚体受体的配体，在维持和呈现干细胞特性方面具有潜在的作用。

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.