| Project/Area Number |
16390502
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Ophthalmology
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| Research Institution | Kyoto Prefectural University of Medicine |
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Principal Investigator |
KINOSHITA Shigeru Kyoto Prefectural University of Medicine, Ophthalmology, Professor, 医学研究科, 教授 (30116024)
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| Co-Investigator(Kenkyū-buntansha) |
KAWASAKI Satoshi Kyoto Prefectural University of Medicine, Ophthalmology, Assistant, 医学研究科, 助手 (60347458)
INATOMI Tsutomu Kyoto Prefectural University of Medicine, Ophthalmology, Assistant, 医学研究科, 助手 (00305583)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2005: ¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 2004: ¥7,100,000 (Direct Cost: ¥7,100,000)
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| Keywords | keratin 12 / corneal epithelium / conjunctival epithelium / transplantation / gene expression / tissue engineering / cytokeratin 12 / amniotic membrane / immunostaining / western blotting / flowcytometry / in situ hybridization |
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| Research Abstract |
The ocular surface is covered by 2 biologically distinct epithelia, the corneal- and the conjunctival epithelium ; the expression of keratin 12 is currently considered a hallmark of cornea-type differentiation. We investigated the biological features of keratin 12-positive cells in human bulbar conjunctival epithelium. Human conjunctival tissues were subjected to investigate the keratin 12 positive cells in conjunctiva by immunostaining, in situ hybridization, western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence-activated cell sorting (FACS). Gene expression profiling of these cells was carried out with introduced amplified fragment length polymorphism (iAFLP). To determine the presence of stem-or progenitor cells, immunostaining and colony-forming assay were performed. Western blotting, RT-PCR revealed that K12 was expressed in conjunctival epithelium. Immunostaining analysis showed that K12 positive cells reside mainly in clusters in conjunctiv
… More
al epithelium. FACS analysis showed that 0.2-1.7% of conjunctival epithelial cells collected from the inferior bulbar conjunctiva were keratin 12-positive. iAFLP analysis revealed that gene expression patterns of these cells were highly similar to that of corneal epithelial cells. p63 and ABCG2 were expressed beneath the keratin 12-positive cells. Some colony-forming cells expressed keratin 12. These results strongly indicated that the keratin 12-positive cells appear to be ectopically-residing, self-maintaining corneal epithelial cells in the conjunctival epithelium. Moreover, we investigated whether conjunctival epithelial cells can be used as alternative source for corneal epithelial cells. We cultivated human conjunctival epithelial cells and made stratified epithelial sheet. This sheet was transplanted onto rabbit cornea under systemic administration of immuno-suppressive agent. The sheet was well-maintained on the zenogeneic rabbit cornea and exhibited several features of corneal epithelial cells such as keratin3/12 expression. These results clearly indicate that human conjunctival epithelial cells can be cultured in vitro and may be used as alternative corneal epithelial cells for the treatment of patients with severe ocular surface diseases. Less
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