腺苷代谢分子CD73经AMPK信号通路调控银屑病Treg细胞及其异常分化的机制研究

Psoriasis is one of the most common immune related skin diseases. The pathogenesis of psoriasis remains unknown and lack of effective treatment. The function imbalance and abnormal differentiation(easily transform to IL-17/Treg cell) of regulatory T cells(Treg) play an important role in psoriasis pathogenesis. Ecto-enzymes CD39/CD73 plays an vital role for their anti-inflammatory effect by Treg cell, and Th17 cell expressed those molecules also. Pre-studies of our group showed that the reduced level of CD73 were significant in peripheral blood Treg cell of psoriasis vulgairs patients. Psoriatic lesions also have CD73+Treg cells involved.The speculation of CD73 deficiency,which may cause the change of psoriatic Treg cell and its differentiation needs further elucidate. Based on the previous work and CD73 related the AMP-activated protein kinase(AMPK), we isolate psoriatic Treg cell in vitro and stimulate it conversion to IL-17/Treg cell. The study also include Imiquimod-Induced psoriasiform mouse model to validate the immunoregulation mechanism of CD73-AMPK pathway in vivo.Combine with the CD73-AMPK pathway intervention experiments, it will help to approve the rationality of pathogenesis hypothesis of “CD73→AMP→AMPK→Treg”, and explore new therapeutic targets for psoriasis.

银屑病是最常见的免疫相关皮肤病，病因不明、治疗乏策。已知其发病与调节性T细胞（Treg）功能紊乱及异常分化相关（极易向IL-17分泌细胞转化，IL-17/Treg）。新近研究表明腺苷代谢分子CD39/CD73在Treg细胞和Th17细胞表面均有表达，并在其免疫功能中发挥重要的作用。课题组发现寻常型银屑病患者外周血Treg细胞CD73表达减少，皮损中亦有CD73+Treg细胞的参与，CD73的缺乏是否导致银屑病Treg细胞功能改变及IL-17/Treg异常分化尚待阐明。本项目旨在前期工作基础上，结合CD73功能相关的磷酸腺苷活化蛋白激酶（AMPK）通路，通过体外分离银屑病外周血Treg细胞并刺激其分化，结合咪喹莫特-银屑病鼠模型，采用CD73-AMPK通路干预实验，探讨“CD73-单磷酸腺苷（AMP）-AMPK-Treg”途径参与银屑病发病的分子机制，为银屑病的治疗提供新的靶点。

银屑病是最为常见的免疫相关皮肤病之一，病因不明、治疗乏策。目前已知其发病与调节性T细胞（Treg）免疫功能紊乱相关。已有研究显示腺苷代谢分子CD39/CD73是Treg细胞发挥抗炎作用的重要途径之一，Th17细胞同样表达该分子，本课题组前期研究发现寻常型银屑病患者外周血Treg细胞表面CD73表达减少，皮损中亦有CD73+Treg细胞的参与，结合AMP激活的蛋白激酶（AMPK）通路，我们推测CD73及其相关信号通路的改变是引起银屑病Treg细胞功能及稳定性改变的原因之一。本课题在前期工作的基础上，体外分离Treg细胞，并结合咪喹莫特-银屑病鼠模型，采用CD73-AMPK通路干预实验，以阐明“CD73→AMP→AMPK→Treg”通路参与银屑病发病的分子机制，以期为银屑病的治疗提供新的靶点。我们首先从银屑病患者和正常人外周血中体外分离Treg细胞、Teff细胞，检测了Treg细胞、Teff细胞共培养后，Teff细胞的增殖、IL-17A和IFN-γ分泌及AMPK/mTOR信号通路的活化，同正常对照相比，银屑病患者Treg细胞免疫抑制功能明显减弱，CD73表达降低，加入银屑病治疗药物甲氨蝶呤（MTX）后，MTX可明显恢复Treg细胞的免疫抑制功能，从而抑制银屑病Teff细胞的过度增殖，同时逆转了CD73的表达减低，上调了AMPK的磷酸化，下调mTOR磷酸化，IL-17A和IFN-γ分泌减少。完成体外实验后，构建咪喹莫特-类银屑病鼠模型，通过HE染色和ELISA检测IL-17、INF-γ表达水平结果表明，模型组有明显的炎症反应，给予MTX治疗后，小鼠银屑病病理状况和炎症反应均有显著改善；当同时给予CD73拮抗剂α,β-甲基ADP和MTX处理后小鼠银屑病病理状况和炎症反应并没有显著改善。甲氨蝶呤（MTX）可通过增加CD73表达/活化AMPK/下调mTOR信号通路，恢复银屑病患者Treg细胞的抑制功能。本研究从CD73-AMPK-mTOR一个全新的角度出发，深入探讨了甲氨蝶呤治疗银屑病的免疫机制，从而为临床中更好地应用甲氨蝶呤治疗银屑病提供了理论基础。