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



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.

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


1. Govindarajan B, Gipson IK. Membrane-tethered mucins have multiple functions on the ocular surface. Exp Eye Res. 2010;90(6):655–63. doi: 10.1016/j.exer.2010.02.014 20223235

2. Ablamowicz AF, Nichols JJ. Ocular Surface Membrane-Associated Mucins. The Ocular Surface. 2016;14(3):331–41. doi: 10.1016/j.jtos.2016.03.003 27154035

3. Rivas L, Blazquez A, Munoz-Negrete FJ, Lopez S, Rebolleda G, Dominguez F, et al. Characterization of epithelial primary culture from human conjunctiva. Archivos de la Sociedad Espanola de Oftalmologia. 2014;89(1):10–6. doi: 10.1016/j.oftal.2013.07.013 24269413

4. Chung So-Hyang, Lee Joon H., Yoon Joo-Heon, Lee Hyung Keun, Seo Kyoung Yul. Multi-layered culture of primary human conjunctival epithelial cells producing MUC5AC. Experimental Eye Research. 2007;85:226–233 17568580

5. Ang L P K, Tan D T H, Seah C J Y, Beuerman R W. The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells. The British Journal of Ophthalmology. 2005;89:748–752 doi: 10.1136/bjo.2004.055046 15923513

6. Chang RS. Continuous subcultivation of epithelial-like cells from normal human tissues. Proceedings of the Society for Experimental Biology and Medicine Society for Experimental Biology and Medicine (New York, NY). 1954;87(2):440–3.

7. Lavappa KS, Macy ML, Shannon JE. Examination of ATCC stocks for HeLa marker chromosomes in human cell lines. Nature. 1976;259(5540):211–3. 1250349

8. Gipson IK, Spurr-Michaud S, Argu¨eso P, Tisdale A, Ng TF, Russo CL. Mucin Gene Expression in Immortalized Human Corneal–Limbal and Conjunctival Epithelial Cell Lines. Investigative Opthalmology & Visual Science. 2003;44(6):2496.

9. O'Sullivan NL, Baylor AE 3rd, Montgomery PC. Development of immortalized rat conjunctival epithelial cell lines: an in vitro model to examine transepithelial antigen delivery. Exp Eye Res. 2007;84(2):323–31. doi: 10.1016/j.exer.2006.10.005 17123516

10. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu C-P, Morin GB, et al. Extension of Life-Span by Introduction of Telomerase into Normal Human Cells. Science. 1998;279(5349):349–52. 9454332

11. Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Current Biology. 1998;8(5):279–82. 9501072

12. Weinberg RA. Telomeres. Bumps on the road to immortality. Nature. 1998;396(6706):23–4. 9817198

13. Matsushita N, Matsushita S, Hirakawa S, Higashiyama S. Doxycycline dependent inducible and reversible RNA interference mediated by a single lentivirus vector. Bioscience, biotechnology, and biochemistry. 2013;77(4):776–81. 23563548

14. Miyoshi H, Blömer U, Takahashi M, Gage FH, Verma IM. Development of a self inactivating lentivirus vector. J Virol. 1998;72(10):8150–57. 9733856

15. Kobayashi T, Shiraishi A, Hara Y, Kadota Y, Yang L, Inoue T, et al. Stromal- epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model. Exp Eye Res. 2015;135:109–17. doi: 10.1016/j.exer.2015.02.009 25682729

16. Ramirez-Miranda A, Nakatsu MN, Zarei-Ghanavati S, Nguyen CV, Deng SX. Keratin 13 is a more specific marker of conjunctival epithelium than keratin 19. Molecular vision. 2011;17:1652–61. 21738394

17. Hayashi Y, Kao WW, Kohno N, Nishihara-Hayashi M, Shiraishi A, Uno T, et al. Expression patterns of sialylated epitope recognized by KL-6 monoclonal antibody in ocular surface epithelium of normals and dry eye patients. Investigative ophthalmology & visual science. 2004;45(7):2212–7.

18. Diebold Y, Calonge M, Enriquez de Salamanca A, Callejo S, Corrales RM, Saez V, et al. Characterization of a spontaneously immortalized cell line (IOBA-NHC) from normal human conjunctiva. Investigative ophthalmology & visual science. 2003;44(10):4263–74.

19. Nicolaissen B Jr., Eidal K, Haaskjold E, Harsem T, Nass O. Outgrowth of cells from human conjunctival explants onto cornea in vitro. Acta ophthalmologica. 1991;69(6):723–30. 1789086

20. Diebold Y, Calonge M, Fernandez N, Lazaro MC, Callejo S, Herreras JM, et al. Characterization of epithelial primary cultures from human conjunctiva. Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie. 1997;235(5):268–76. 9176674

21. Sharif NA, Crider JY, Griffin BW, Davis TL, Howe WE. Pharmacological analysis of mast cell mediator and neurotransmitter receptors coupled to adenylate cyclase and phospholipase C on immunocytochemically-defined human conjunctival epithelial cells. Journal of ocular pharmacology and therapeutics: the official journal of the Association for Ocular Pharmacology and Therapeutics. 1997;13(4):321–36.

22. Sharif NA, Xu SX, Magnino PE, Pang IH. Human conjunctival epithelial cells express histamine-1 receptors coupled to phosphoinositide turnover and intracellular calcium mobilization: role in ocular allergic and inflammatory diseases. Exp Eye Res. 1996;63(2):169–78. 8983974

23. Hollingsworth MA, Swanson BJ. Mucins in cancer: protection and control of the cell surface. Nature reviews Cancer. 2004;4(1):45–60. 14681689

24. Rheinwald J. G., Hahn W. C., Ramsey M. R., Wu J. Y., Guo Z., Tsao H., De Luca M., Catricala C. and O'Toole K. M. A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status. Mol. Cell. Biol. 2002;22:5157–5172. doi: 10.1128/MCB.22.14.5157-5172.2002 12077343

25. Marko Christina K., Menon Balaraj B., Chen Gang, Whitsett Jeffrey A., Clevers Hans, Gipson Ilene K. Spdef Null Mice Lack Conjunctival Goblet Cells and Provide a Model of Dry Eye. The American Journal of Pathology. 2013;183(1):35–48 doi: 10.1016/j.ajpath.2013.03.017 23665202

26. Marko Christina Kaiser, Tisdale Ann S., Spurr-Michaud Sandra, Evans Christopher, Gipson Ilene K. The Ocular Surface Phenotype of Muc5ac and Muc5b Null Mice. Invest. Ophthalmol. Vis. Sci. 2014;55(1):291–300. doi: 10.1167/iovs.13-13194 24327612

27. Zhang Yujin, Lam Oliver, Nguyen Minh-Thanh T., Ng Gracia, Pear Warren S., Ai Walden, Wang I-Jong, Kao Winston W.-Y., Liu Chia-Yang. Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity. Development 2013;140(3):594–605 doi: 10.1242/dev.082842 23293291

28. McCauley Heather A., Liu Chia-Yang, Attia Aria C., Wikenheiser-Brokamp Kathryn A., Zhang Yujin, Whitsett Jeffrey A., Guasch Géraldine. TGFβ signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium. Development 2014;141:4628–4639 doi: 10.1242/dev.117804 25377551

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