中华皮肤科杂志 ›› 2015, Vol. 48 ›› Issue (6): 391-394.

• 论著 • 上一篇    下一篇

中波紫外线对体外培养HaCaT细胞MAPK信号通路的影响

徐倩倩1,陈旭2,李莉3,徐松2,丁兰4,张云1,鞠梅5,李新宇6,施辛1,陈崑6,顾恒2   

  1. 1. 苏州大学附属第二医院
    2. 中国医学科学院北京协和医学院皮肤病研究所
    3. 中国医学科学院皮肤病医院
    4. 苏州大学附属第二医院皮肤科
    5. 南京 中国医学科学院北京协和医学院皮肤病医院
    6. 南京 中国医学科学院北京协和医学院皮肤病研究所
  • 收稿日期:2014-09-24 修回日期:2014-11-12 出版日期:2015-06-15 发布日期:2015-06-03
  • 通讯作者: 陈旭 E-mail:doctor_chx@126.com

Effects of ultraviolet B on the MAPK signaling pathway in cultured HaCaT cells in vitro

  • Received:2014-09-24 Revised:2014-11-12 Online:2015-06-15 Published:2015-06-03

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摘要：

目的 探讨中波紫外线照射后不同时间点体外培养的HaCaT细胞MAPK信号通路受调控效应。 方法 1.5、4.5、7.5、10、20、30、50 mJ/cm2中波紫外线照射HaCaT细胞后8 h，对照组细胞除不进行UVB照射外，其他处理同各剂量UVB组，蛋白免疫印迹法测定ERK1/2、JNK和p38蛋白表达及磷酸化水平；50 mJ/cm2中波紫外线照射HaCaT细胞，蛋白免疫印迹法测定照射后2、4、8、12 h 4个时间点，细胞ERK1/2、JNK和p38蛋白表达及磷酸化水平。使用Quantity One软件计算目的条带吸光度，以相应蛋白条带的吸光度值与GAPDH蛋白内参条带吸光度值的比值表示相对蛋白含量。 结果 7.5、10、20、30、50 mJ/cm2 UVB照射HaCaT细胞后8 h，ERK1/2、JNK磷酸化水平明显上调（P < 0.05），20、30、50 mJ/cm2 UVB照射HaCaT细胞后p38磷酸化水平上调显著（P < 0.05），且都在50 mJ/cm2照射后效应最为显著（P < 0.05）。50 mJ/cm2 UVB照射HaCaT细胞后8 h，ERK1/2磷酸化产物水平出现上调，在处理后的12 h，与对照组（10.277 ± 0.320）比较，差异有统计学意义（44.844 ± 2.023，P < 0.05），而p38和JNK磷酸化产物水平在照射后2 h即开始上调（P < 0.05），4、8、12 h，p38和JNK与对照相比，UVB照射后虽存在着显著地磷酸化水平差异（P < 0.05），但随时间推移JNK磷酸化产物水平这种上调的趋势逐渐弱化（P < 0.05）。 结论 在50 mJ/cm2中波紫外线照射后的HaCaT细胞中，ERK1/2、p38、JNK这3种MAPK信号通路产生了活化效应，具有一定的时间效应差异。

Abstract:

Xu Qianqian*, Chen Xu, Li Li, Xu Song, Ding Lan, Zhang Yun, Ju Mei, Li Xinyu, Shi Xin, Chen Kun, Gu Heng. *Department of Dermatology, Second Affiliated Hospital of Soochow University, Suzhou 215004, China Corresponding authors: Chen Xu, Email: doctor_chx@126.com; Shi Xin, Email: shx9@163.com 【Abstract】 Objective To explore the regulatory effects of ultraviolet B （UVB） radiation on the mitogen-activated protein kinase （MAPK） signaling pathway in cultured HaCaT cells in vitro at different post-radiation time points. Methods Some cultured HaCaT cells were classified into several groups to be exposed to UVB of 1.5, 4.5, 7.5,10, 20, 30 and 50 mJ/cm2 for 1, 3, 5, 7, 15, 20 and 34 seconds respectively, or UVB of 50 mJ/cm2 for 34 seconds, or remain untreated （control group）. Western blot was performed to determine the total and phosphorylation levels of ERK1/2, JNK and p38 in HaCaT cells at 8 hours after exposure to 1.5, 4.5, 7.5,10, 20, 30 and 50 mJ/cm2 UVB, as well as at 2, 4, 8 and 12 hours after exposure to 50 mJ/cm2 UVB. Quantity One software was used to measure the absorbance value of protein bands, and protein levels were expressed as the absorbance ratio of target bands to glyceraldehyde-3-phosphate dehydrogenase （GAPDH）. Statistical analysis was carried out by using one-way analysis of variance, least significant difference （LSD）-t test and multivariate analysis of variance. Results Compared with the control group, there was a significant increase at 8 hours in the phosphorylation levels of ERK 1/2 and JNK in HaCaT cells after exposure to 7.5, 10, 20, 30 and 50 mJ/cm2 UVB （all P < 0.05）, and in the phosphorylation level of p38 after exposure to 20, 30 and 50 mJ/cm2 UVB （all P < 0.05）, with the strongest increase in phosphorylation levels of ERK 1/2, JNK and p38 all observed in HaCaT cells exposed to 50 mJ/cm2 UVB （all P < 0.05）. After exposure to 50 mJ/cm2 UVB, the phosphorylation level of ERK1/2 began to increase in HaCaT cells at 8 hours, and significantly increased at 12 hours （44.844 ± 2.023 vs. 10.277 ± 0.320, P < 0.05） compared with the control group; however, the phosphorylation levels of p38 and JNK increased in HaCaT cells as early as 2 hours after exposure （both P < 0.05）, and remained significantly higher compared with the control group at 4, 8 and 12 hours （all P < 0.05）, while the increasing trend in phosphorylated JNK level weakened over time （P < 0.05）. Conclusions The ERK1/2, JNK and p38 MAPK signaling pathways were activated in HaCaT cells after exposure to 50 mJ/cm2 UVB, and the degree of their activation varied over time.