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Koji Kitazawa, Satoshi Kawasaki, Katsuhiko Shinomiya, Akira Matsuda, Toshinari Funaki, Mina Nakatsukasa, Kenta Yamazaki, Nobuyuki Ebihara, Akira Murakami, Shigeru Kinoshita; Establishment Of Immortalized Corneal And Conjunctival Epithelial Cells Lacking The Functional Tacstd2 Gene. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1820.
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© ARVO (1962-2015); The Authors (2016-present)
Gelatinous drop-like corneal dystrophy (GDLD) is a single-gene disorder caused by biallelic mutations within the tumor-associated calcium signal transducer 2 (TACSTD2) gene and is characterized by bilateral corneal amyloidosis. Currently, the only effective treatment for this disease is replacement of the cornea, however, recurrence of the disease can occur within several years after the replacement. Thus, the development of novel effective treatments for GDLD is a need that is still unmet. The purpose of this present study was to establish immortalized corneal and conjunctival epithelial cells lacking the functional TACSTD2 gene.
A small piece of corneal or conjunctival tissue was obtained from two GDLD patients (both bearing biallelic p.Gln118X) at the time of surgery. The epithelial cells were enzymatically separated and dissociated from the obtained tissues and introduced with the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes by the use of lentiviral vector. Next, population doubling (PD) analysis, transepithelial resistance analysis, immunostaining, and western blot analysis of the cells were performed to investigate the life span of the cells and the reasonability of using the cells as an in vitro model for GDLD.
The corneal and conjunctival epithelial cells introduced with the SV40 large T antigen and hTERT genes continued to proliferate even after the cumulative number of PDs exceeded 40, while the cells without such gene transfection stopped proliferating when the cumulative number of PDs reached 18. Transepithelial resistance of the transfected cells was significantly lower compared to immortalized normal conjunctival epithelial cells. The expression level of the claudin 1 and 7 proteins was significantly lower compared to the immortalized normal conjunctival epithelial cells, consistent with the what we observed in the cornea of GDLD patients in our previous report.
The results of this study show that we successfully established immortalized corneal and conjunctival epithelial cells derived from GDLD patients. We hope that our established immortalized cells will contribute to the future development of effective treatments for GDLD.
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