INTRODUCTION: Primary cultures of @entity1 corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for @entity1 and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by @entity2235 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties. METHODS: Primary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using @entity11185 solution, an @entity5292 antibiotic. To ensure that the immortalized cell lines express SV40 T and @entity16914 , a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis. RESULTS: These cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) @entity876 protein were significantly decreased in the immortalized cell lines while the expression of total @entity876 protein was constant. In addition, expression of @entity3544 ( @entity994 ), a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, @entity16914 ( @entity16914 ), was expressed at equal levels in the immortalized cells and primary HCE cells. DISCUSSION: Taken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method.

Immortalization of XXXX corneal epithelial cells using @entity2235 large T antigen and cell characterization.

@entity1 :: ('9606', 'Species') :: ['human'] @entity16914 :: ('3850', 'Gene') :: ['CK-3', 'cytokeratin-3'] @entity11185 :: ('MESH:C010680', 'Chemical') :: ['G418'] @entity2235 :: ('10633', 'Species') :: ['simian virus 40'] @entity3544 :: ('644914', 'Gene') :: ['p21'] @entity5292 :: ('D000617', 'Chemical') :: ['aminoglycoside'] @entity876 :: ('7157', 'Gene') :: ['p53'] @entity994 :: ('1026', 'Gene') :: ['cip1']
@entity1 :: (9606,Species) :: ['human']
