中华皮肤科杂志 ›› 2016, Vol. 49 ›› Issue (2): 88-92.

• 论著 • 上一篇    下一篇

携带HPV11基因组的HaCaT细胞APOBEC3s表达及α干扰素的调控

王永芳1,李新宇2,宋莎莎2,孙洋3   

  1. 1. 中国医学科学院北京协和医学院皮肤病研究所
    2. 南京 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
  • 收稿日期:2015-07-16 修回日期:2015-09-24 出版日期:2016-02-15 发布日期:2016-02-04
  • 通讯作者: 李新宇 E-mail:xinyusli609@163.com
  • 基金资助:

    协和青年基金资助和中央高校基本科研业务费专项资金

Expressions of APOBEC3s in HaCaT keratinocytes carrying the genome of human papillomavirus type 11 and regulatory effects of interferon-alpha on the expressions

  • Received:2015-07-16 Revised:2015-09-24 Online:2016-02-15 Published:2016-02-04

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摘要：

目的 探讨携带HPV11基因组的HaCaT细胞（HPV11.HaCaT）中载脂蛋白B信使RNA编辑酶催化多肽样蛋白3家族（APOBEC3s，A3s）mRNA表达、主要A3蛋白在细胞内的表达、分布，以及外源性α干扰素对其调节。 方法 qRT-PCR检测HPV11.HaCaT细胞中A3A、A3B、A3C和A3H mRNA的基础表达水平，并与正常HaCaT细胞作比较；分别用不同浓度的重组人IFN-α2b（rhIFN-α2b）作用于HaCaT、HPV11.HaCaT、Hela细胞6、24、48 h后，qRT-PCR检测A3A、A3B、A3C和A3H mRNA表达水平。细胞免疫荧光染色法观察rhIFN-α2b刺激6 h时，A3A蛋白在3种细胞中的表达和分布情况。 结果 HPV11.HaCaT细胞内A3A、A3B、A3C mRNA表达量均明显高于正常HaCaT细胞（P < 0.05）。在不同浓度rhIFN-α2b刺激下，3种细胞4种A3成员mRNA表达趋势存在差异，其中A3A升高最为明显。与各自正常对照组相比，106 IU/ml rhIFN-α2b刺激6 h时3种细胞的A3A mRNA表达明显升高，差异有统计学意义（HaCaT细胞35.77 ± 5.01比1.00 ± 0.05，P < 0.05；HPV11.HaCaT细胞15.34 ± 2.14比0.99 ± 0.01，P < 0.05；Hela 24.60 ± 5.45比0.97±0.03，P < 0.05），而A3B、A3C和A3H mRNA相对表达量均低于10倍。经106 IU/ml rhIFN-α2b处理6 h后，3种细胞胞质和胞核中均可见到A3A蛋白的阳性染色增加。 结论 HPV11进入HaCaT细胞后，可激活A3s（尤其是A3A）免疫系统。α干扰素诱导A3A的高表达可能是其发挥免疫调控功能的机制之一。

Abstract:

Wang Yongfang, Li Xinyu, Song Shasha, Sun Yang Pharmacal Research Laboratory, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding author: Li Xinyu, Email: xinyusli609@163.com 【Abstract】 Objective To investigate mRNA expressions of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 family （APOBEC3s or A3s） as well as expressions and subcellular distribution of major A3 proteins in HaCaT keratinocytes carrying the genome of human papillomavirus type 11 （HPV11.HaCaT）, and to evaluate regulatory effects of exogenous interferon-alpha （IFN-α） on the expressions of A3s. Methods The basal levels of A3A, A3B, A3C and A3H mRNA expressions were measured by real-time fluorescence-based quantitative PCR （qRT-PCR） in HPV11.HaCaT cells and normal HaCaT cells. Cultured HaCaT, HPV11.HaCaT and Hela cells were treated with recombinant human IFN-α 2b （rhIFN-α2b） at concentrations of 104, 105 and 106 IU/ml for 6, 24 and 48 hours separately, and those receiving no treatment served as the normal control groups. Then, qRT-PCR was performed to measure mRNA expressions of A3A, A3B, A3C and A3H in these cells. Immunofluorescence staining was conducted to observe the expression and distribution of A3A protein in cells after the treatment with rhIFN-α2b for 6 hours. Results As qRT-PCR showed, the basal levels of A3A, A3B and A3C mRNA expressions were all significantly higher in HPV11.HaCaT cells than in normal HaCaT cells （all P < 0.05）. After stimulation, the mRNA expressions of the four A3 members increased to different extents with the increase in rhIFN-α2b concentrations, and the increase in A3A mRNA was the most significant. Compared with corresponding normal control groups, the mRNA expression of A3A was significantly increased in HaCaT cells （35.77 ± 5.01 vs. 1.00 ± 0.05, P < 0.05）, HPV11.HaCaT cells （15.34 ± 2.14 vs. 0.99 ± 0.01, P < 0.05） and Hela cells （24.60 ± 5.45 vs. 0.97 ± 0.03, P < 0.05） after the treatment with rhIFN-α2b at 106 IU/ml for 6 hours, while the increase in A3B, A3C and A3H mRNA expressions was no more than 9-fold in these cell lines after that. Enhanced staining for A3A was observed in nuclei and cytoplasm of the 3 cell lines after the treatment with rhIFN-α2b at 106 IU/ml for 6 hours. Conclusions HPV11 transfected into HaCaT cells can activate intracellular A3s, especially A3A. IFN-α may play an immunoregulatory role by inducing high levels of A3A expression.