不同剂量中波紫外线照射对角质形成细胞增殖活力和自噬体表达影响的研究

【摘要】 目的 观察HaCaT细胞和原代角质形成细胞接受不同剂量中波紫外线（UVB）照射后细胞增殖活力和自噬体表达水平的变化，并初步评估增殖活力损伤程度和自噬体表达水平之间的相关性。 方法 两种细胞各分为5组，对照组、5、10、20、40 mJ/cm2 UVB照射组，照射结束后12 h进行MTT实验或单丹（磺）酰戊二胺（MDC）染色，全波长酶标仪下读取各孔A值，倒置荧光显微镜下随机选取视野，计数每视野下自噬体表达阴性细胞和阳性细胞数目。 结果 经不同剂量UVB照射后，HaCaT细胞的增殖活力（A值）较原代角质形成细胞下降更明显，其中HaCaT细胞10、20、40 mJ/cm2照射组（A值分别为1.367 ± 0.035、1.173 ± 0.034、0.873 ± 0.025）两两之间以及与对照组（1.519 ± 0.022）之间差异均有统计学意义（P < 0.01）；原代角质形成细胞仅10、20、40 mJ/cm2 UVB照射组（A值分别为0.782 ± 0.012、0.773 ± 0.021、0.725 ± 0.031）与对照组（0.887 ± 0.035）之间差异有统计学意义（P < 0.05）。5、10、20 mJ/cm2 UVB照射后两种细胞MDC染色，自噬体表达阳性的细胞比率均出现增加，但照射量至40 mJ/cm2时则出现下降，以原代角质形成细胞下降更明显，其中HaCaT细胞10 mJ/cm2和20 mJ/cm2 UVB照射组自噬体表达阳性率（分别为22.69% ± 2.15%、28.10% ± 2.92%）较对照组（10.18% ± 1.50%）有显著上升，而40 mJ/cm2组自噬体表达上升幅度出现下降（趋势卡方检验χ2 = 27.48，P < 0.01）；原代角质形成细胞对照组、5、10、20 mJ/cm2组间自噬体阳性率变化不大，但40 mJ/cm2组表达出现明显抑制（趋势卡方检验χ2 = 6.86，P < 0.01）。 结论 UVB对HaCaT细胞和原代角质形成细胞增殖活力的损伤均有剂量依赖性，原代角质形成细胞更耐受UVB损伤；5、10、20 mJ/cm2 UVB照射能促进HaCaT细胞自噬体表达增加，且有剂量依赖性，而对原代角质形成细胞自噬体表达水平未产生显著影响；40 mJ/cm2 UVB照射后HaCaT细胞自噬体表达有下降趋势，而显著抑制原代角质形成细胞自噬体水平的表达。 【关键词】 角质形成细胞； 自噬体； 紫外线

Effect of different doses of ultraviolet B on the proliferation of and autophagosome formation in keratinocytes

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