甲氨蝶呤通过调控Treg细胞治疗银屑病的药理机制研究

Many researches have confirmed the regulatory effect of MTX on Treg cells in inflammatory diseases. This project aims to explore the role of above function in the treatment of psoriasis. We will analyze the number of Treg cells as well as the expression of its membrane labeling molecules (Foxp3 / CTLA - 4) and secreted inflammatory mediators (IL - 10 / TGF-β) in different types of psoriasis, and their connection with the disease period and its severity; Then we will establish psoriasis like dermatitis on mice ,both the mice models and patients with psoriasis vulgar will be treated with MTX in order to observe the effect of MTX on activation of Treg cells and clarify the internal signaling pathway both in skin lesions and the blood circulation; In addition, we will establish a method for Treg and Teff cells co-cultur technique to explore the effect of MTX on the function of Treg cells, meanwhile, we will reveal the molecular basis of MTX on regulation of Treg cells by using siRNA to knock down signal pathway molecules or its agonist/antiagonist agents；Finally, we will observe the treatment effect of Treg cells or the optimal agonist/antiagonist of signal pathway molecules in vivi on psoriasis like dermatitis on mice. This project will play an important role to reveal the immunological mechanism of psoriasis, clarification of the regulation MTX on Treg cells will provide a new thought of treatment related to immunology, and construct an important bridge toward to the research of new drug translational medicine in psoriasis.

大量研究已证实甲氨蝶呤（MTX）对于炎症性疾病Treg细胞的调控作用，本项目旨在银屑病中探索上述调控作用及分子机制。首先分析Treg细胞及标记分子（Foxp3/CTLA-4）和分泌因子（IL-10/TGF-β）的表达和银屑病类型，病期及严重性的关联；以稳定期斑块状银屑病和咪喹莫特诱导的小鼠银屑病样皮炎为研究对象，揭示MTX对皮损及循环Treg细胞的调控，探讨调控的信号途径变化；另外，我们建立Treg和Teff细胞共培养技术，研究MTX对Treg细胞功能的影响，运用siRNA技术或干预芯片筛选的信号通路关键分子，验证MTX调控Treg细胞的信号途径。最后，我们观察体外诱导的Treg细胞或信号通路激动/阻断剂对银屑病样皮炎的治疗效应。本项目对于揭示银屑病新的免疫学机制有重要意义，阐明MTX对Treg细胞的调控将为银屑病治疗提供了新的思路，为针对银屑病新药转化医学研究搭建了桥梁。

1本项目揭示MTX治疗银屑病的药理学机制：（1）研究发现MTX治疗可以有效下调血浆中腺苷水平，上调外周血Tregs细胞，下调外周血MDSCs细胞。运用IMQ诱导的银屑病鼠模型发现：模型鼠脾脏及皮损中Tregs下调并存在功能缺陷，MDSCs存在上调， MTX通过调控Tregs的数量和膜分子的表达治疗银屑病，对MDSCs有着负调控作用。在机制方面，发现腺苷受体A2aR在IMQ诱导的银屑病鼠模型中表达下降，其表达和疾病严重程度PASI呈负相关性，说明A2aR可能是MTX药理学机制的关键靶点，进一步靶向A2aR构建条件性表皮敲除小鼠，经IMQ诱导出更加严重的银屑病样表型，具体表现为表皮增厚明显，鳞屑较多。以上结果初步揭示了腺苷-A2aR通路是MTX治疗银屑病的内在药理学机制。（2）运用临床采集的MTX治疗有效/无效的银屑病患者的生物学样本，运用全外显子测序的方法筛选MTX疗效相关的SNPs位点并进行验证，初步获得银屑病MTX疗效相关的预测位点，发现ABCB1 rs1045642 TT基因型多态性可以预测中国银屑病人群银屑病对MTX治疗的临床反应，以上结果为我国MTX在银屑病治疗领域的个体化精准用药奠定理论基础。（3）进一步探寻银屑病新的血清学治疗靶点和创新型的药物，积极进行临床科研转化研究。相关研究成果发表第一或通讯作者论文7篇，其中SCI论文6篇。作为课题负责人获得2018年度国家重点研发计划1项和湖南省科技厅重点研发计划1项，以微信小程序为载体，构建了“询肤知详”，实现了银屑病的筛查，准确率达到94%，申报专利1项，作为主要完成人获得“湖南省自然科学一等奖”。项目负责人获得中华预防医学会皮肤病与性病学委员、湖南省医学会银屑病学组委员/秘书、中华医学会皮肤病与性病学分会免疫学组 委员、银屑病防治教育专项基金委员会委员等学术任职。