5-Azac诱导急性移植物抗宿主病免疫低反应的甲基化研究

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

目的 建立小鼠急性移植物抗宿主病（a GVHD）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a GVHD免疫低反应后的甲基化变化,探讨5-Azac对小鼠a GVHD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 m L/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（Me DIP-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 Me DIP-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a GVHD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a GVHD的免疫低反应。

Objective To analyse the change of DNA methylation with 5-Azac injection in acute graft-versus-host dis-ease （aGVHD） mouse model, which received allogeneic bone marrow transplantation, and explore the immunomodulatory ef- fects 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-Azae experi- mental group. At 1-7, 14, 21 and 28-day after transplantion, 5-Azae 0.25 mg/kg （0.3 mUtime） 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 MeDIP- seq method was selected to detect methylation changes in mice, and the differential DNA methylation in the biological path- ways 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 down- regulated 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. Conclu- sion 5-Azae can effectively induce immune hyporesponsiveness post aGVHD by changing the gene methylation status.