关键词: 黑素细胞, 角蛋白细胞, 成纤维细胞, 细胞增殖, 蓝光, 黑素合成

Abstract: 【Abstract】 Objective To preliminarily investigate the effect of blue light on the biological activity of human skin keratinocytes, fibroblasts and melanocytes. Methods Discarded foreskin tissues were collected from 10 healthy children aged from 3 to 12 years after circumcision surgery in the First Affiliated Hospital of Kunming Medical University from June 2021 to December 2021. After epidermis-dermis separation, selective culture was performed to isolate keratinocytes, fibroblasts, and melanocytes. According to the pre-experiment results, the above three types of cells were irradiated with 440 - 450 nm blue light at doses of 0, 5, 10, 20, 30, and 40 J/cm2, and then continued to be cultured for 0, 6, 24, and 48 hours. Cell counting kit 8 （CCK8） assay was performed to evaluate cellular proliferative activity at each time point, enzyme-linked immunosorbent assay （ELISA） to detect levels of interleukin （IL）-18, IL-33, nerve growth factor （NGF）, and granulocyte-macrophage colony-stimulating factor （GM-CSF） secreted by keratinocytes, as well as levels of IL-33 and keratinocyte growth factor （KGF） secreted by fibroblasts, NaOH lysis method to determine melanin synthesis rates in melanocytes, and Western blot analysis to determine the relative expression of tyrosinase （TYR）, tyrosine-related protease 1 （TRP-1） and dopachrome isomerase （DCT） in melanocytes. Two-way analysis of variance was used to analyze group effects, time effects and interaction effects. Results After irradiation with blue light, the cellular proliferative activity significantly differed among different doses of blue light irradiation groups and different time points in keratinocytes （Ftime = 516.20, Fdose = 421.20, Finteraction = 25.05, all P < 0.003）, fibroblasts （Ftime = 129.30, Fdose = 477.80, Finteraction = 10.91, all P < 0.003）, and melanocytes （Ftime = 77.61, Fdose = 138.70, Finteraction = 3.50, all P < 0.003）; immediately after irradiation, the proliferative activity of keratinocytes and fibroblasts was significantly lower in the 20 - 40 J/cm2 blue light group than in the 0 J/cm2 blue light group （all P < 0.003）, and the proliferative activity of melanocytes was significantly higher in the 5 J/cm2 blue light group than in the 0 J/cm2 blue light group （P < 0.003）; the proliferative activity of the 3 types of cells showed decreasing trends with the increase of blue light irradiation doses and culture time. ELISA showed that the concentrations of IL-18, IL-33, NGF, and GM-CSF secreted by keratinocytes, as well as the concentrations of IL-33 and KGF secreted by fibroblasts, tended to increase with the increase of blue light irradiation doses and culture time. The melanin synthesis rates in melanocytes significantly differed among different doses of blue light irradiation groups and different time points （Ftime = 833.50, Fdose = 249.40, Finteraction = 81.38, all P < 0.003）; during 0 - 24 hours after blue light irradiation, the melanin synthesis rates tended to increase with the increase of blue light irradiation doses and time; during 24 - 48 hours, the melanin synthesis rates showed decreasing trends with the increase of blue light irradiation doses and culture time compared with that at 24 hours after irradiation; 24 hours after irradiation, the melanin synthesis rates were significantly higher in the 5, 10, 20, 30 and 40 J/cm2 blue light groups （159.50% ± 10.88%, 218.76% ± 8.49%, 333.72% ± 7.72%, 393.29% ± 6.00%, 427.21% ± 8.39%, respectively） than in the 0 J/cm2 blue light group （102.29% ± 6.57%, all P < 0.003）. The relative expression of TYR （Ftime = 67.94, Fdose = 28.99, Finteraction = 3.71, all P < 0.003）, TRP-1 （Ftime = 21.73, Fdose = 8.38, both P < 0.003） and DCT （Ftime = 34.51, Fdose = 11.79, both P < 0.003） in melanocytes significantly differed among different doses of blue light irradiation groups and different time points, and tended to increase with the increase of blue light irradiation doses and culture time. Conclusion Blue light irradiation at doses of 5 - 40 J/cm2 could inhibit the proliferative activity of human skin keratinocytes, fibroblasts, and melanocytes, and the inhibitory effect tended to increase with the increase of blue light irradiation doses, except an enhancing effect on the proliferative activity of melanocytes observed immediately after irradiation with blue light at 5 J/cm2; additionally, blue light irradiation at 5 - 40 J/cm2 could enhance the expression of melanin synthesis-related enzymes in melanocytes, and increase the melanin synthesis rate in melanocytes over a short period of time.

Key words: Melanocytes, Keratinocytes, Fibroblasts, Cell proliferation, Blue light, Synthesis of melanin