Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line


Autoři: Arisa Mitani aff001;  Takeshi Kobayashi aff002;  Yasuhito Hayashi aff001;  Natsuki Matsushita aff003;  Sachi Matsushita aff004;  Saori Nakao aff001;  Naoko Takahira aff001;  Atsushi Shiraishi aff001;  Yuichi Ohashi aff001
Působiště autorů: Department of Ophthalmology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, Japan aff001;  Department of Ophthalmology and Regenerative Medicine, Ehime University Graduate School of Medicine, Toon, Ehime, Japan aff002;  Division of Laboratory Animal Research, Aichi Medical University, Nagakute, Aichi, Japan aff003;  Translational Research Center, Ehime University Hospital, Toon, Ehime, Japan aff004;  Department of Biochemistry, Aichi Gakuin University School of Dentistry, Nagoya, Japan aff005
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222454

Souhrn

Purpose

To present the properties of a newly developed immortalized human conjunctival epithelial cell (iHCjEC) line.

Methods

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.

Results

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.

Conclusion

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.

Klíčová slova:

Biology and life sciences – Cell biology – Cellular types – Animal cells – Epithelial cells – Cell physiology – Cell immortalization – Anatomy – Biological tissue – Epithelium – Developmental biology – Cell differentiation – Biochemistry – Proteins – Collagens – Mucin – Genetics – Gene expression – Molecular biology – Molecular biology techniques – Artificial gene amplification and extension – Polymerase chain reaction – Medicine and health sciences – Research and analysis methods – Biological cultures – Cell cultures


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PLOS One


2019 Číslo 9

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