天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (2): 173-177.doi: 10.11958/20150153

• 实验研究 • 上一篇    下一篇

5-Azac 诱导急性移植物抗宿主病免疫低反应的甲基化研究

张晓宁， 赵玉霞， 王建海， 苗绪红， 李克秋， 李光△   

  1. 天津医科大学基础医学院生物学教研室 （邮编 300070）
  • 收稿日期:2015-09-09 修回日期:2015-09-23 出版日期:2016-02-15 发布日期:2016-02-15
  • 通讯作者: △通讯作者 E-mail： lig@tmu.edu.cn E-mail:zhangxiaoning@tmu.edu.cn
  • 基金资助:
    国家高技术研究发展计划 （863计划） 资助项目 （2012AA021003）； 国家自然科学基金资助项目 （21177091）

Analysis of DNA methylation with 5-Azac induced immune hyporesponsiveness following acute graft-versus-host disease

ZHANG Xiaoning， ZHAO Yuxia， WANG Jianhai， MIAO Xuhong， LI Keqiu， LI Guang△   

  1. Department of Biology, Basic Medical College, Tianjin Medical University, Tianjin 300070, China
  • Received:2015-09-09 Revised:2015-09-23 Published:2016-02-15 Online:2016-02-15
  • Contact: △Corresponding Author E-mail： lig@tmu.edu.cn E-mail:zhangxiaoning@tmu.edu.cn

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摘要： 目的 建立小鼠急性移植物抗宿主病 （aGVHD） 模型， 检测 DNA 甲基化转移酶抑制剂 5-氮杂胞苷 （5-aza⁃ cytidine， 5-Azac）诱导 aGVHD 免疫低反应后的甲基化变化， 探讨 5-Azac 对小鼠 aGVHD 的免疫调节作用。方法选择雄性 C57BL/6（H-2b）与雌性 BALB/c（H-2d）小鼠分别作为异基因移植供、 受体建立移植物抗宿主病小鼠模型。 BABL/c 受体按照体质量相近进行配对， 分为移植对照组和 5-Azac 实验组。5-Azac 实验组于移植后 1~7、 14、 21、 28 d 尾静脉注射 5-Azac 0.25 mg/kg（0.3 mL/只）； 移植对照组尾静脉注射生理盐水 0.3 mL/只。提取 3 只移植对照组和 3 只 5-Azac 实验组小鼠的外周血 DNA， 分别等量混匀， 采用甲基化 DNA 免疫共沉淀测序 （MeDIP-seq） 的方法检测甲基化变化， 筛选差异甲基化基因， 并对其功能及生物学通路进行分析。结果 5-Azac 实验组小鼠的生存时间延长，排斥反应减弱， 成功诱导移植物抗宿主病免疫低反应状态。2 组小鼠 DNA MeDIP-seq 结果对比显示： 5-Azac 实验组启动子区存在 369 个差异甲基化基因， 其中上调 239 个、 下调 130 个； 外显子区存在 184 个差异甲基化基因， 其中上调 113 个、 下调 71 个。利用 KEGG （Kyoto Encyclopedia of Genes and Genomes） 数据库对差异甲基化基因分析， 结果显示其主要参与 10 个免疫学信号通路， 其中 TGF-β、 GSK-3β、 SYK、 PI3K、 NFAT、 CD28、 α4β7 与 aGVHD 的发生发展密切相关。结论 5-Azac 可以通过改变基因的甲基化状态有效诱导 aGVHD 的免疫低反应。

关键词: 移植物抗宿主病, 甲基化, 5-氮杂胞苷, MeDIP-seq, 免疫低反应

Abstract: Objective To analyse the change of DNA methylation with 5-Azac injection in acute graft-versus-host disease (aGVHD) mouse model, which received allogeneic bone marrow transplantation, and explore the immunomodulatory effects of 5-Azac. Methods Male C57BL/6 (H-2b） and female BALB/c (H-2d) mice were selected as donor and recipient of complete allotransplantation. BABL/c mice were divided into two groups, transplantation control group and 5-Azac experi⁃ mental group. At 1-7, 14, 21 and 28-day after transplantion, 5-Azac 0.25 mg/kg (0.3 mL/time) was injected by tail vein in experimental group, while the control group were injected with sterile water 0.3 mL/time. Peripheral blood DNA samples were collected from three control mice and three experimental mice, then mixed with equal amount respectively. The MeDIPseq method was selected to detect methylation changes in mice, and the differential DNA methylation in the biological pathways was analyzed. Results The survival time was prolonged, and the rejection reaction was decreased in 5-Azac experi⁃ mental group, which suggested immune hyporesponsiveness post aGVHD. The MeDIP-seq result showed that 369 different DNA methylation located in the promoter regions, including 239 up-regulated genes and 130 down-regulated genes. There were 184 differential DNA methylation genes located in the exon regions, including 113 up-regulated genes and 71 downregulated genes. Differential DNA methylation genes involved in 10 immunological signaling pathways, respectively. Among them, TGF-β, GSK-3β, SYK, PI3K, NFAT, CD28 and α4β7 were closely related to the development of aGVHD. Conclusion 5-Azac can effectively induce immune hyporesponsiveness post aGVHD by changing the gene methylation status.

Key words: graft vs host disease, methylation, 5-azacytidine, MeDIP-seq, immune hyporesponsiveness