Document Detail


Establishment of human corneal epithelial cells stably expressing human connexin43.
MedLine Citation:
PMID:  19799899     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Kazuhiro Kimura; Shinichiro Teranishi; Teruo Nishida
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-09-30
Journal Detail:
Title:  Experimental eye research     Volume:  90     ISSN:  1096-0007     ISO Abbreviation:  Exp. Eye Res.     Publication Date:  2010 Jan 
Date Detail:
Created Date:  2009-12-04     Completed Date:  2010-01-15     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370707     Medline TA:  Exp Eye Res     Country:  England    
Other Details:
Languages:  eng     Pagination:  4-9     Citation Subset:  IM    
Affiliation:
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:
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:
0/Connexin 43; 0/Recombinant Fusion Proteins; 0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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