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目的:观察人永生化角质形成细胞系HaCaT细胞接受不同剂量长波紫外线(UVA)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:将HaCaT细胞分为6组,分别为10 J/cm~2UVA对照组、10 J/cm~2 UVA照射组、25 J/cm~2 UVA对照组、25 J/cm~2 UVA照射组、50 J/cm~2 UVA对照组及50 J/cm~2 UVA照射组。照射结束后即刻进行噻唑蓝(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UVA照射后,HaCaT细胞的增殖活力(A值)下降,呈剂量相关性。其中10、25及50 J/cm~2 UVA照射组(A值分别为1.179±0.007、0.791±0.015、0.522±0.046)两两之间以及与各自对照组(1.370±0.007、1.254±0.012、1.177±0.009)间差异均有统计学意义(P均<0.01);10、25及50 J/cm~2 UVA照射后,HaCa T细胞MDC染色结果示自噬体表达阳性的细胞比率增加,且呈剂量相关性。50 J/cm~2 UVA照射组自噬体表达阳性率较其对照组显著上升(χ~2=11,P<0.01)。结论:10~50 J/cm~2 UVA照射后,HaCaT细胞瞬时增殖活力降低及自噬体表达增加,且呈剂量依赖性。
OBJECTIVE: To observe the changes of proliferation activity and autophagosome expression of human immortalized keratinocyte cell line HaCaT after UVA irradiation at different doses, and to evaluate the relationship between the degree of proliferative activity damage and the expression level of autophagosome Correlation. Methods: HaCaT cells were divided into 6 groups: 10 J / cm ~ 2 UVA control group, 10 J / cm ~ 2 UVA irradiation group, 25 J / cm ~ 2 UVA control group and 25 J / , 50 J / cm ~ 2 UVA control group and 50 J / cm ~ 2 UVA irradiation group. Thiazolyl blue (MTT) test or monoamidocaproic acid (MDC) staining was performed immediately after the irradiation. A value of each well was read under a full-wavelength microplate reader. The field of view was randomly selected under an inverted fluorescence microscope and counted per field of view The number of negative cells and positive cells under autophagosome expression. Results: The proliferation activity (A value) of HaCaT cells decreased with dose UVA irradiation in a dose-dependent manner. (A values were 1.179 ± 0.007,0.791 ± 0.015 and 0.522 ± 0.046 respectively) between the groups of 10, 25 and 50 J / cm ~ 2 UVA irradiation and between the control groups (1.370 ± 0.007,1.254 ± 0.012,1.177 ± 0.009) (all P <0.01). The results of MDC staining of HaCa T cells after 10, 25 and 50 J / cm 2 UVA irradiation showed that the percentage of cells expressing positive autophagosomes increased Dose-related. The positive rate of autophagosome expression in 50 J / cm ~ 2 UVA irradiation group was significantly higher than that in control group (χ ~ 2 = 11, P <0.01). CONCLUSION: The transient proliferation activity of HaCaT cells and the increase of autophagosomes after exposure to 10 ~ 50 J / cm ~ 2 UVA are dose-dependent.