Abstract: HUANG Dan, REN Fa-liang, CHEN Xu, CHEN Kun, GU Heng. Department of Physiotherapy, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: CHEN Kun, Email: kunchen181@aliyun.com; GU Heng, Email: guheng@aliyun.com 【Abstract】 Objective To observe the changes in proliferative activity of and autophagosome formation in human HaCaT keratinocytes and primary keratinocytes after different doses of ultraviolet B （UVB） radiation, and to assess the potential relationship between proliferation impairment and autophagosome formation. Methods Both cultured HaCaT cells and primary keratinocytes from human foreskin were irradiated with different doses （5, 10, 20 and 40 mJ/cm2） of UVB. Those receiving no irradiation served as the control. After additional 12-hour culture, methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate the proliferative activity of cells, monodansylcadaverin （MDC） staining to detect autophagosomes in cells. The number of autophagosome-positive or -negative cells was counted using inverted fluorescence microscopy. Results UVB radiation induced a significant decrease in the proliferation of keratinocytes, especially in that of HaCaT cells. The proliferative activity expressed as the absorbance value at 490 nm was significantly lower in HaCaT cells （1.367 ± 0.035, 1.173 ± 0.034 and 0.873 ± 0.025 vs. 1.519 ± 0.022, all P < 0.01） and primary keratinocytes （0.782 ± 0.012, 0.773 ± 0.021 and 0.725 ± 0.031 vs. 0.887 ± 0.035, all P < 0.05） irradiated with UVB of 10, 20 and 40 mJ/cm2 than in the unirradiated control cells. Significant differences were also observed in the proliferative activity among HaCaT cells irradiated with UVB of 10, 20 and 40 mJ/cm2. The proportion of autophagosome-positive cells was increased after irradiation with UVB of 5, 10 and 20 mJ/cm2, but decreased after irradiation with UVB of 40 mJ/cm2 in keratinocytes, especially in the primary keratinocytes. In detail, the proportion of autophagosome-positive cells was 22.69% ± 2.15%, 28.10% ± 2.92% and 22.92% ± 2.61% in HaCaT cells irradiated with UVB of 10, 20 and 40 mJ/cm2 respectively, significantly higher than that in the unirradiated cells （10.18% ± 1.50%, chi-square test for trends: χ2 = 27.48, P < 0.01）. No significant changes were observed in the proportion of autophagosome-positive cells in primary keratinocytes after irradiation with UVB of 5, 10 and 20 mJ/cm2, but a marked decrease was found after irradiation with UVB of 40 mJ/cm2 compared with unirradiated keratinocytes （chi-square test for trends: χ2 = 6.86, P < 0.01）. Conclusions UVB radiation（10 - 40 mJ/cm2） decelerates the proliferation of HaCaT cells and primary keratinocytes in a dose-dependent manner, and primary keratinocytes seem to be more resistant to UVB damage than HaCaT cells. Low to moderate doses （5 - 20 mJ/cm2） of UVB promote autophagosome formation in HaCaT cells in a dose-dependent manner, and exert no significant influence on that in primary keratinocytes; however, UVB of 40 mJ/cm2 suppresses autophagosome formation in keratinocytes, especially in primary keratinocytes. 【Key words】 Keratinocytes; Autophagy; Ultraviolet rays

Key words: keratinocytes, Autophagy