ESTABLISHMENT OF AGED HUMAN DERMAL FIBROBLASTS BY ULTRAVIOLET IRRADIATION

Introduction: Photoaging is a degenerative condition that leads to skin fragility, loss of function and cosmetic dissatisfaction. The senescence of human dermal fibroblasts (HDFs) plays vital roles in the pathogenesis of skin aging. Therefore, we aim to develop a senescent HDFs model with an optimized acute ultraviolet (UV) irradiation protocol for investigating mechanism and developing novel treatment intervention for photoaging. Materials and methods: HDFs were isolated from human abdominal skin and the expression of some fibroblast-specific markers was evaluated by flow cytometry. HDFs were then divided into three groups: normal (non-UV), UV1, and UV2. The irradiation dosages were UVB 780 mJ/cm2 + UVA 480 mJ/cm2 for 10 mins in group UV1, and UVB 1170 mJ/cm2 + UVA 720 mJ/cm2 for 15 mins in group UV2. The HDFs were then assessed with some markers for aging. The cell morphology and size were observed by microscopy. SA-β-galactosidase expression of cells was stained by SA-β-Gal kit and measured using Image J software. The differentiation potential of HDFs into chondrocytes, osteocytes, and adipocytes was checked by inducible medium. Cell proliferation was stained by Alamar blue assay and measured by spectrophotometer. Gene expression of collagen 1, collagen 3, MMP-3, p16, and p21 were quantified by real-time RT‒PCR. Results: The results showed that isolated cells exposed spindle-shape morphology, and highly expressed with fibroblast markers such as CD 90 (63%), Vimentin (85.55%), and S100A4 (61.17%). After UV irradiation, in the UV-treated groups, the cell appearance became flattened and larger. Cell sizes were significantly increased in UV-irradiated HDFs (p<0.05). The integrated density of SA-β-gal signals was higher in the UV groups than in the normal group (p<0.05). The time for fibroblasts to differentiate into adipocytes and chondrocytes was longer in both UV groups compared to normal HDFs. The division ability of cells significantly decreased in both UV groups at 72 hours and was maintained until 8 days (p<0.001), with a lower proliferation rate in the UV2 group. Moreover, UV light increased the mRNA expression of MMP-3, p16, and p21 and decreased the expression of collagen 1 and 3 in the UV2 group (p<0.05). Conclusion: These results demonstrated that the single irradiation dose of UVB 1170 mJ/cm2 + UVA 720 mJ/cm2 for 15 mins has successfully established the HDF senescence model.