| Project/Area Number |
15591879
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 | TOKYO DENTAL COLLEGE |
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Principal Investigator |
SHIMAZAKI Jun Tokyo Dental College, Department of Dentistry, Associate Professor, 歯学部, 助教授 (40170930)
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| Co-Investigator(Kenkyū-buntansha) |
TSUBOTA Kazuo Tokyo Dental College, Cornea center, Chief of Research, 角膜センター, 教授(研究部長) (40163878)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Amniotic membrane / HLA-G / corneal coniunctival epitherial cells / Anti-inflammatory / immune response / Cytotoxic assay / 細胞障害性試験 / IFN-γ |
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| Research Abstract |
PURPOSE. To investigate the expression and function of HLA-G in limbal and conjunctival epithelial cells when expanded on amniotic membrane(AM). METHODS. A conjunctival epithelial cell line (CCL20.2) was used to screen mRNA expression of HLA molecules when cultured with or without amniotic membrane. Expression of HLA-G mRNA in primary cultured limbal and conjunctival epithelial cells was measured by RT-PCR and real-time PCR. Immunocytochemical staining and western blot analysis was done to confirm HLA-G protein expression. The HLA class I negative K-562 human erythroleukaemia cell line was transfected with limbal epithelium-derived HLA-G mRNA for functional analysis using an NK cell-lysis assay.
RESULTS. Microarray analysis revealed increased expression of HLA-G, and decreased expression of other HLA genes when CCL20.2 cells were cultured on AM rather than plastic. Limbal and conjunctival epithelial cells were also shown to express HLA-G by RT-PCR, immunocytology and western blots. Semi-quantitative real-time PCR showed a significant increase in HLA-G expression in conjunctival epithelial cells when cultured on AM. Although expression of HLA-G in limbal epithelial cells did not change with AM substrate alone, a more than 2-fold greater upregulation was observed following the addition of IFN-γ when cultured on AM. NK cell-induced cytolysis of K-562 cells was slightly inhibited by transfection of HLA-G.
CONCLUSION. Conjuctival and corneal epithelial cells express functional HLA-G when expanded ex vivo, which is enhanced by using an AM substrate and by IFN-γ stimulation.
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