Establishment and characterization of immortalized human gingival keratinocyte cell lines.
OBJECTIVE
Primary human keratinocytes are used to analyze the properties of the oral epithelium and the early stages of oral bacterial infections. In vitro, these cells are characterized by their short life span and restricted availability. Approaches for culturing these cells will end after approximately 6-10 passages as a result of entry into apoptosis. For this reason, it is important to generate cell lines suitable for obtaining an unlimited source of cells. Therefore, the aim of the present study was to generate gingival keratinocyte cell lines and to compare their in vitro behaviour with those of primary human gingival keratinocytes.


METHODS
Primary human gingival keratinocytes were immortalized with a combination of the human papilloma virus onkoproteins E6 and E7. The pattern of the cytokeratins, involucrin and filaggrin was investigated by intracellular staining using flow cytometry. This method allows quantitative analysis of the expression of a variety of intracellular or extracellular markers.


RESULTS
The immortalized cell lines showed many morphological similarities, expressing a cytokeratin pattern that is comparable with that of primary gingival keratinocytes. Furthermore, they developed transepithelial electrical resistance, which is a marker for the generation of tight junctions. These results indicate that the cells might be able to act as an epithelial barrier, reflecting the reaction of primary human cells.


CONCLUSIONS
The establishment of a continuous line of human gingival epithelial cells with functional characteristics of the epithelial barrier provides a valuable in vitro model for using to study the early steps of gingival/periodontal infections.
Gröger
S
S
Sabine Gröger, Schlangenzahl 14, Giessen 35394, Germany. Sabine.E.Groeger@dentist.med.uni-giesseen.de
Michel
J
J
Meyle
J
J
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D062445:Claudin-1; D017097:Electric Impedance; D004846:Epithelial Attachment / Q000166:cytology* / Q000502:physiology; D000091344:Filaggrin Proteins; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005881:Gingiva / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007381:Intermediate Filament Proteins / Q000096:biosynthesis; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D007633:Keratins / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins / Q000235:genetics; D011498:Protein Precursors / Q000096:biosynthesis; D019108:Tight Junctions; D014162:Transfection
C568108:CLDN1 protein, human; D062445:Claudin-1; C118860:E6 protein, Human papillomavirus type 6; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D008565:Membrane Proteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011498:Protein Precursors; C032253:involucrin; D007633:Keratins