| Budget Amount *help |
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2003: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 2002: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Research Abstract |
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.
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