不同剂量中波紫外线对HaCaT细胞p62及自噬体形成关键基因Beclin-1、Atg12和Atg3的调控效应研究

中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (4): 233-236.

不同剂量中波紫外线对HaCaT细胞p62及自噬体形成关键基因Beclin-1、Atg12和Atg3的调控效应研究

金慧¹,陈旭²,徐松²,曹蕾³,黄丹⁴,鞠梅⁵,陈崑⁶,李新宇⁶,周之海¹,顾恒²

1. 天津医科大学总医院皮肤科
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研究所
4. 中国医学科学院皮肤病研究所理疗科
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:2013-07-03 修回日期:2014-01-28 出版日期:2014-04-15 发布日期:2014-04-01
通讯作者: 陈旭 E-mail:doctor_chx@126.com
基金资助:
国家自然科学基金面上项目;江苏省基础研究计划（自然科学基金）面上项目;2011年度高等学校博士学科点专项科研基金;2013年度北京协和医学院协和青年科研基金

Regulatory effects of different doses of ultraviolet B on the expressions of p62, Beclin-1, Atg12 and Atg3 in HaCaT cells

Received:2013-07-03 Revised:2014-01-28 Online:2014-04-15 Published:2014-04-01

摘要/Abstract

摘要： 【摘要】目的探讨不同剂量中波紫外线（UVB）照射培养的HaCaT细胞后不同时间点对p62、Beclin-1、Atg12和Atg3蛋白表达水平的调控效应。方法使用4.5、10和50 mJ/cm2 UVB照射HaCaT细胞，照射后加入新鲜培养基继续培养4 h和12 h，同时设相同处理但不照射UVB的细胞为对照细胞。另设观察组，在照射后立即（包括对照细胞），使用含蛋白酶抑制剂E64D（10 μg/L）和胃酶抑素（10 μg/L）的培养基孵育4 h和12 h，以阻断对p62的降解。使用Western印迹法测定HaCaT细胞p62、Beclin-1、Atg12和Atg3蛋白的表达水平。结果 50 mJ/cm2 UVB照射HaCaT细胞后4 h，p62表达水平（p62与内参的比值为0.473 ± 0.022）较对照细胞（0.246 ± 0.038）升高（t = 15.27，P < 0.05）；阻断溶酶体后，细胞新生p62的水平（0.445 ± 0.035）与阻断溶酶体的对照（0.244 ± 0.016）相比，仍为上调（t = 7.62，P < 0.05）。在4.5 mJ/cm2 UVB照射细胞后12 h，与对照（0.254 ± 0.035）相比，HaCaT细胞p62表达上调（0.497 ± 0.047，t = 22.89，P < 0.05）；阻断溶酶体后，与阻断溶酶体的对照（0.257 ± 0.025）相比，p62表达水平仍然上调（0.548 ± 0.051，t = 17.42，P < 0.05）。4.5 mJ/cm2和50 mJ/cm2 UVB照射后4 h和12 h，对照细胞和照射细胞间的Beclin-1、Atg12和Atg3的表达差异均无统计学意义（P > 0.05），阻止溶酶体对自噬体内容物的降解也未能发现Beclin-1、Atg12和Atg3的表达差异（P > 0.05）。结论 p62表达在不同剂量UVB照射和照射后不同时间存在差异调控，并且这种调控效应与自噬体形成可能没有生物学联系。

关键词: 紫外线, 角蛋白细胞, 原癌基因蛋白质类, Beclin-1, Atg12, Atg3

Abstract: Jin Hui *, Chen Xu, Xu Song, Cao Lei, Huang Dan, Ju Mei, Chen Kun, Li Xinyu, Zhou Zhihai, Gu Heng. *Department of Dermatology, Tianjin Medical University General Hospital, Tianjin 300052, China Corresponding authors: Chen Xu, Email: doctor_chx@126.com; Zhou Zhihai, Email: zhouzh65@sohu.com 【Abstract】 Objective To evaluate the regulatory effects of different doses of ultraviolet B （UVB） radiation on the expressions of p62, Beclin-1, Atg12 and Atg3 proteins in the human keratinocyte cell line HaCaT. Methods Cultured HaCaT cells were divided into several groups to be irradiated with three doses of UVB （4.5, 10 and 50 mJ/cm2） immediately followed by additional culture with or without the lysosomal protease inhibitors E64D of 10 μg/L and pepstatin of 10 μg/L, both of which were used to block p62 degradation, for 4 and 12 hours respectively. Those HaCaT cells receiving the same treatment but no irradiation served as the control. Subsequently, Western blot was performed to determine the expression levels of p62, Beclin-1, Atg12 and Atg3 proteins in these cells. The protein expression level was represented as the ratio of the product of absorbance value and area of the electrophoretic bands of target proteins to that of β-tubulin. Paired t test was performed to compare the expression levels of these proteins among different groups. Results After irradiation with UVB of 50 mJ/cm2, the expression level of p62 was significantly upregulated at 4 hours in unblocked HaCaT cells compared with unblocked unirradiated cells （0.473 ± 0.022 vs. 0.246 ± 0.038, t = 15.27, P < 0.05）, and in blocked HaCaT cells compared with blocked unirradiated cells （0.445 ± 0.035 vs. 0.244 ± 0.016, t = 7.62，P < 0.05）. Increased p62 expression was also observed in unblocked and blocked HaCaT cells at 12 hours after irradiation with UVB of 4.5 mJ/cm2 compared with the corresponding unirradiated cells （unblocked cells: 0.497 ± 0.047 vs. 0.254 ± 0.035, t = 22.89, P < 0.05; blocked cells: 0.548 ± 0.051 vs. 0.257 ± 0.025, t = 17.42, P < 0.05）. No significant differences were noted in the protein expression levels of Beclin-1, Atg12 or Atg3 between unblocked or blocked HaCaT cells and corresponding unirradiated cells at 4 or 12 hours after irradiation with UVB at 4.5 or 50 mJ/cm2 （all P > 0.05）. Conclusions The expression level of p62 is different at different time points after different doses of UVB radiation, and there seems no biological association between UVB radiation and autophagosome formation.

Key words: Ultraviolet rays, Keratinocytes, Proto-oncogene proteins, Beclin-1, Atg12, Atg3

金慧陈旭徐松曹蕾黄丹鞠梅陈崑李新宇周之海顾恒. 不同剂量中波紫外线对HaCaT细胞p62及自噬体形成关键基因Beclin-1、Atg12和Atg3的调控效应研究[J]. 中华皮肤科杂志, 2014, 47(4): 233-236.

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