Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line.
Antigens, Viral, Tumor
/ metabolism
Cell Culture Techniques
/ methods
Cell Line
/ metabolism
Cell Line, Transformed
/ metabolism
Cells, Cultured
Conjunctiva
/ metabolism
Doxycycline
/ metabolism
Epithelial Cells
/ metabolism
Gene Expression
/ genetics
Gene Expression Regulation
/ genetics
Humans
RNA, Messenger
/ genetics
Simian virus 40
/ metabolism
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2019
2019
Historique:
received:
12
04
2019
accepted:
29
08
2019
entrez:
12
9
2019
pubmed:
12
9
2019
medline:
3
4
2020
Statut:
epublish
Résumé
To present the properties of a newly developed immortalized human conjunctival epithelial cell (iHCjEC) line. iHCjECs were developed to induce Simian Virus 40 large T-antigen (SV40LT) by incorporating lentivirus in a tetracycline (Tet)-regulated gene-expression system into primary cultures of human conjunctival epithelial cells. The population doubling time and morphology of the iHCjECs were analyzed. The expressions of CK13, CK19, CK12, and MUC1, MUC4, MUC16, and MUC5AC were determined by real time PCR and immunohistochemically under different culture conditions. The organotypic culture model in which iHCjECs were cultured on rabbit conjunctival fibroblast-embedded collagen gel was used to characterize the iHCjECs. The iHCjECs cultured with doxycycline (Dox) continued to proliferate for at least 20 passages and had a cobblestone-like appearance. The expressions of CK13 and CK19 but not CK12 were detected in the iHCjECs, and the expression of CK13 increased in culture media lacking Dox (Dox-). The expressions of MUC1, MUC4, MUC16, and MUC5AC were detected in iHCjECs, and a relatively strong immunostaining of MUC5AC was detected with Dox(-) added 5% FBS. Stratified iHCjECs were observed in organotypic culture at 5 days. The iHCjECs had high proliferation rates and abilities to control the differentiation potency to control the expression of SV40 LT-antigen with Tet-regulated gene-expression system. They are able to express the mucin gene repertoire of their native epithelia. The iHCjECs can be a useful experimental cell line to study conjunctival epithelial cell characteristics and for pathophysiological and toxicological studies.
Identifiants
pubmed: 31509592
doi: 10.1371/journal.pone.0222454
pii: PONE-D-19-10471
pmc: PMC6738650
doi:
Substances chimiques
Antigens, Viral, Tumor
0
RNA, Messenger
0
Doxycycline
N12000U13O
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0222454Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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