| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
The ocular surface consists of two types of epithelium, corneal epithelium (CE) and conjunctival epithelium (CJE), yet their biological characteristics and responsibility for neovascularization are different. In order to treat a variety of ocular surface diseases with distraction of histological structure and stem cell deficiency, epithelial transplantation is an effective procedure for clinical recovery. As an initial step for the development of a new medical treatment, it is important to understand the epithelial differentiation and the mechanisms for avascularity. This project investigated the expression profiling of 36 genes related to neovascularization of corneal, limbal, and conjunctival epithelium, and it was carried out using introduced amplified fragment length polymorphism (iAFLP). Thrombospondin 1(TSP1) mRNA expressed by basal cells in the limbus and cornea was significantly higher than that expressed in the conjunctiva. TSP1 protein is also expressed in the basal region of
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the cornea and limbus, but not in the conjunctiva. Immunohistochemistry revealed that TSP-1 was strongly expressed in the basal region of the CE, but its expression was faint in limbal epithelium (LE) and absent in CJE. There was little or no labeling in the CJE. High levels of TSP-1 are present only just below the CE, and its unique distribution may be related to corneal avascularity and integrity. We have developed a new process for ocular surface reconstruction using cultivated corneal (CCE) and oral mucosal epithelial sheet transplantation (COMET), and reported on their clinical efficacy. However, corneal neovascularization after transplantations were different in each sheet, suggesting characteristic differences between CCE and COMET in regards to angiogenesis. We examined the expression of angiogenesis-related factors thrombospondin-1 (TSP-1), pigment epithelium derived factor (PEDF), endostatin, angiostatin, vascular endothelial growth factor (VEGF), Fms-like tyrosine kinase 1 (Flt-1), kinase insert domain receptor (KDR), and basic fibroblast growth factor (bFGF). We used Western blot analysis to confirm the factors that were immunohistochemically different in CCE and COE. The immunohistochemical staining intensity of TSP-1 was higher in CCE than in COE, and Western blot analysis showed that the expression of TSP-1 was significantly higher in CCE than in COE (p<0.05). PEDF and endostatin stained moderately stronger in CCE than in COE. There was no obvious immunohistochemical difference between CCE and COE with respect to angiostatin, VEGF, Flt-1, KDR, and bFGF. In comparison with CCE, COE showed a decreased expression of anti-angiogenic factors TSP-1, PEDF, and endostatin. These different expressions may relate to the superficial peripheral neovascularization encountered after COE transplantation. These results may contribute to the development of additional procedures for ocular surface reconstruction and prevention of corneal stem cell deficiency disorders. Less
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