| Establishment of human corneal epithelial cells stably expressing human connexin43. | |
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MedLine Citation:
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PMID: 19799899 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Corneal epithelial cells communicate with each other through gap junctions. Whereas this property is retained in corneal epithelial cells in primary culture, it is often lost in immortalized epithelial cells. However, the life span of primary cultured corneal epithelial cells is short and the availability of human tissue for their preparation is limited. To examine the role of the gap-junction protein connexin43 (Cx43) in human corneal epithelial cells, we set out to establish an immortal human corneal epithelial cell line that stably expresses this protein. An expression vector encoding human Cx43 fused to enhanced green fluorescent protein (EGFP) was constructed and introduced by transfection into SV40-immortalized human corneal epithelial (HCE) cells. Stable transfectants were isolated by selection with the antibiotic G418. The expression and localization of the Cx43-EGFP fusion protein were examined by immunoblot analysis and fluorescence microscopy, respectively, and gap-junctional intercellular communication was monitored on the basis of dye coupling. HCE cells stably expressing Cx43-EGFP manifested intercellular dye transfer, whereas those stably expressing EGFP alone did not. Cx43-EGFP localized to the interfaces of neighboring cells. Stable expression of Cx43-EGFP in HCE cells did not affect the expression of keratins 3 and 12, which is a characteristic of corneal epithelial cells, but it did inhibit cell proliferation. We have established an HCE cell line that stably expresses human Cx43 and forms functional gap junctions. These cells may prove useful for studies of the role of gap junctions in the human corneal epithelium. |
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Authors:
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Kazuhiro Kimura; Shinichiro Teranishi; Teruo Nishida |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2009-09-30 |
Journal Detail:
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Title: Experimental eye research Volume: 90 ISSN: 1096-0007 ISO Abbreviation: Exp. Eye Res. Publication Date: 2010 Jan |
Date Detail:
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Created Date: 2009-12-04 Completed Date: 2010-01-15 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0370707 Medline TA: Exp Eye Res Country: England |
Other Details:
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Languages: eng Pagination: 4-9 Citation Subset: IM |
Affiliation:
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Department of Ocular Pathophysiology, Yamaguchi University, Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. k.kimura@yamaguchi-u.ac.jp |
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| MeSH Terms | |
Descriptor/Qualifier:
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Cell Line Cell Proliferation Cloning, Molecular Connexin 43 / genetics* Electrophoresis, Polyacrylamide Gel Epithelium, Corneal / cytology, metabolism* Gap Junctions / physiology* Gene Expression Regulation / physiology* Genetic Vectors Green Fluorescent Proteins / genetics Humans Immunoblotting Microscopy, Fluorescence Plasmids Recombinant Fusion Proteins / genetics Simian virus 40 / genetics Transfection |
| Chemical | |
Reg. No./Substance:
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0/Connexin 43; 0/Recombinant Fusion Proteins; 0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins |
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