Establishment of skin photoaging model and preliminary mechanism of senescence in keratinocytes cell line HaCaT

Abstract

Objective To establish an efficient skin photoaging model in HaCaT cells and investigate the possible signaling pathways of photoaging of skin. Methods After HaCaT cells were exposed to UVB at 30 mJ/cm2, cell proliferation and cell cycle was detected by CCK-8 assay and flow cytometry. The percentages of positive cells were detected by SA-β-gal staining. The expression of p53, p21 and p16 at mRNA and protein levels were detected by qRT-PCR and Western blotting respectively. The subcellular localization of p21 was elucidated by immunofluorescence assay. Results Exposure of UVB at 30 mJ/cm2 resulted in significantly decreased proliferation (P < 0.01), increased proportion of cells at G2 phage (P < 0.05), and more positive cells to SA-β-gal (P < 0.05) when compared with normal control cells. The results of qRT-PCR and Western blotting showed that UVB exposure up-regulated the expression levels of p53 and p16, and down-regulated that of p21 in HaCaT cells (P < 0.05, P < 0.01). Immunofluorescence assay indicated obvious nuclear localization of p21 in the HaCaT cells after UVB exposure. Conclusion UVB-exposure at 30 mJ/cm2 successfully establish skin photoaging model in HaCaT cells, which may be due to senescence induced by increasing nuclear localization of p21 and activating p16-dependent pathway.

Keywords

• photoaging
• cell model
• p53
• p21
• p16