Effect of different doses of ultraviolet B on the proliferation of and autophagosome formation in keratinocytes / 中华皮肤科杂志

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 (5,10,20and 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 trendsx2 =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 trendsx2 =6.86,P ＜ 0.01).Conclusions UVB radiation (10-40 mJ/cm2) decelerates the proliferation of HaCaT cells and primary keratinocytes in a dosedependent 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.