MK/T-1, an immortalized fibroblast cell line derived usingcultures of mouse corneal stroma

[摘要] Purpose: Immortalized cell lines representing fibroblast cells fromcorneal stroma would facilitate studies of corneal cell biology andinjury response.Methods: Primary cultures of cells derived from mouse corneal stromawere transfected with a human telomerase reverse transcriptase (hTERT)expression construct to maximize chances of cellular immortalization. Aresulting cell line was analyzed for telomerase activity, cell growthcharacteristics, senescence and gene expression patterns. Specificresponses to transforming growth factor b (TGF-b) were alsoanalyzed.Results: An immortalized cell line was derived and was named MK/T-1.MK/T-1 cells show no signs of cellular senescence or transformation atover 100 passages. Telomerase activity was significantly higher inMK/T-1 cells as compared to the parental cell cultures. However,relative telomere length (RTL) in the MK/T-1 and parental cells was notsignificantly different. Senescence associated b-galactosidase(SA-b-Gal) activity was not detected in late passage MK/T-1cells while the parental cells had already upregulated SA-b-Gal athigh levels by passage 9. The MK/T-1 cells express vimentin, tubulin,lumican, mimecan, decorin and collagen I, but not keratocan. Exposure ofthe MK/T-1 cells to TGF-b induces the expression of smooth musclea-actin (ASMA), the activation of MAP Kinase (p38-MAPK) andmorphological changes consistent with cytoskeletal reorganization.Conclusions: MK/T-1 cells represent an immortalized fibroblast cellline derived using cultures from corneal stroma cell preparations.Expression of hTERT may contribute to immortalization of the MK/T-1cells by a mechanism other than increases in RTL. MK/T-1 cells may be auseful model in which to study the responses of corneal fibroblast cellsto cytokines and other diverse environmental factors in vitro.

[学科分类] 生物化学/生物物理