去甲基化作用诱导的NF-κB表达上调和Foxp3基因位点去甲基化在调节性γδ T细胞生成中的作用

急性移植物抗宿主病（aGVHD）是异基因造血干细胞移植（HSCT）的常见死亡原因。前期研究中我们首次发现体内含量极少的调节性γδT细胞（γδTreg）体外诱导扩增后过继输注能有效防治aGVHD，首次表明地西他滨能高效诱导该细胞生成从而保障了细胞来源。但其诱导机制不明确，我们提出NF-κB表达上调和Foxp3基因去甲基化可能起关键作用。本项目拟通过芯片、免疫印迹和甲基化测序验证地西他滨在上调NF-κB表达和Foxp3基因去甲基化中的作用，进一步应用荧光素酶报告系统阐明去甲基化的Foxp3基因通过增强顺式作用元件活性促进Foxp3表达的分子机制，并应用分子生物学手段从细胞和分子水平揭示不同表达量的NF-κB直接结合Foxp3基因顺式作用元件的能力及机制，进而应用ChIP on Chip技术寻找调控Foxp3表达的新靶点，为γδTreg研究和应用所需细胞数量提供保障，为aGVHD防治提供新思路。

Acute graft-versus-host disease（aGVHD） is a common cause of death afer allogeneic hematopoietic stem cell transplantation. In our previous study we found for the first time that adoptive transfer of regulatory γδ T（γδ Treg）cells play vital roles in aGVHD prevention and treatment but its quantity in vivo is limited. We also found that decitabine can increase the induction of γδ Treg cells for the sake of providing enough cell sources.But the mechanisms of cell induction remains unkown.We hypothesize that decitabine increases the induction of γδTregs via NF-κB up-regulation and Foxp3 gene demethylation.Based on these,the current research will demonstrate the role of decitabine in NF-κB up-regulation and Foxp3 gene loci demethylaiton by chip, western blot and methylation sequence.We will further clarify the molecular mechanisms for enhancing the cis-acting elements' function by the demethylated Foxp3 gene loci to promote Foxp3 expression with the help of dual luciferase report gene assay system. Then we demonstrate the binding ability of different NF-κB expression levels with cis-acting elements and its mechanisms from cellular and molecular levels by various experiments. Finally we will find novel targets for regulation Foxp3 gene expression via ChIP on Chip. The research can lay the foundation for providing enough cell quantity for further research and application. Moreover, it will also privide new clues to aGHVD prevention and treatment in clinic.