Abstract:

Objective To investigate the role of ultraviolet B (UVB) in induction of senescence of human skin fibroblasts and explore its mechanism. Methods Primary human skin fibroblasts were isolated and cultured by tissue explants adherent method, and UVB irradiation dose for induction of senescence of skin fibroblasts for subsequent experiment was determined through observation of cell morphology and β-galactosidase (β-gal) cell senescence staining after irradiation with different doses of UVB. Cells and supernatant of cell culture were collected at different time points (12, 24, 48 and 72 h) after irradiation with established doses of UVB. The expression of marker P16 protein in induced senescent cells was detected by Western blotting, and expression of typeⅠcollagen, type III collagen, matrix metalloproteinase (MMP)-1 and MMP-3 of senescent cells was detected by ELISA. Results Human skin fibroblasts exhibited typical morphology of cell senescence after irradiation with 40 mJ/cm² UVB, the percent of cells with positive staining with β-gal for cell senescence was (88.75±5.32)%, and the established irradiation dose for induction of cell senescence was 40 mJ/cm². Western blotting analysis revealed that the expression of P16 in induced  senescent skin fibroblasts increased with time of UVB irradiation. It was indicated by ELISA that the expression of typeⅠcollagen and type Ⅲ collagen significantly decreased with time of UVB irradiation in induced senescent cells, while the expression of MMP1 and MMP3 significantly increased with time of UVB irradiation. Conclusion Senescence of human skin fibroblasts can be induced by UVB irradiation, and the mechanism may relate to the decrease of collagen synthesis and increase of collagen degradation.

Key words: human skin fibroblasts, senescence, collagen, matrix metalloproteinase, ultraviolet B