Demonstration of P-selectin expression and potential function in human corneal epithelial cells
| Autor: | Rebecca A. Dawson, Damien G. Harkin, Peter J. Gillies, Jennifer Walshe, Sally-Anne Stephenson, Neil A. Richardson |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Immunocytochemistry Blotting Western Gene Expression Cell Separation Limbus Corneae Real-Time Polymerase Chain Reaction Corneal limbus 03 medical and health sciences Cellular and Molecular Neuroscience 0302 clinical medicine Keratin medicine Humans Cells Cultured Corneal epithelium chemistry.chemical_classification Membrane Glycoproteins Epithelium Corneal Fibroblasts Molecular biology Immunohistochemistry eye diseases Sensory Systems Blot Ophthalmology P-Selectin 030104 developmental biology medicine.anatomical_structure chemistry Cell culture 030221 ophthalmology & optometry sense organs Immortalised cell line Immunostaining Biomarkers |
| Zdroj: | Experimental eye research. 176 |
| ISSN: | 1096-0007 |
| Popis: | In response to an unexpected observation of apparent localisation by immunocytochemistry, we have investigated the potential expression and function of P-selectin (CD62P) in human corneal epithelial cells. The SV40 immortalised cell line, HCE-T (validated by STR profiling), along with multiple donor corneal-limbal tissue samples, were examined for P-selectin expression using a combination of immunocytochemistry, Western blotting, RT-PCR and immunohistochemistry. Potential expression of the major ligand for P-selectin (P-selectin glycoprotein ligand-1; PSGL-1; CD162) was also examined by immunocytochemistry and RT-PCR. A selective inhibitor of P-selectin-PSGL-1 binding (KF38789) was subsequently tested for effects on HCE-T cells using a cell culture gap-closure assay. HCE-T cells as well as primary epithelial cultures derived from donor corneal-limbal tissue, displayed positive immunostaining for P-selectin. Staining was particularly evident at cell-cell boundaries and at the outer edge of expanding epithelial islands. P-selectin expression was confirmed by Western blotting and RT-PCR (validated by product sequencing), as well as by immunohistochemistry performed on serial sections of corneal-limbal tissue stained for P-selectin, keratin 3 and p63. PSGL-1 was detected by RT-PCR and immunocytochemistry in both corneal epithelial cells as well as human limbal fibroblasts (HLF). KF38789 (5 μM) significantly reduced closure of a 500-μm gap between confluent sheets of HCE-T cells over an 8-hr period (by ∼40%, p |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect