树突状细胞Shp2磷酸酶调控过敏性哮喘作用机制研究

Environmental allergens stimulating dendritic cells (DCs) are the key process of asthma and the molecular mechanism of DC regulating asthma is a new hot spot for the drug screening. Non-receptor phosphatase Shp2 is an important signal transduction molecule and its activation depends on the membrane receptor anchoring and combination. Gab1, an adaptor protein with binding sites of Shp2, is a new-found susceptibility factor of asthma. Our previous study has found that Gab1 mediates asthma process by regulating CCR7 signal. However, the downstream phosphorylation of signal transducer is unclear. Preliminary experiments showed that Shp2 can be recruited by Gab1 and activated in DCs under the stimulation of allergens, which implies that Shp2 is a key downstream component involved in the regulation of DC function. As a continuation of the previous work, we constructed the mice of Shp2 specific-deletion in DCs (CD11-Cre: Shp2F/F) and phenotype analysis suggested that loss of Shp2 relieved the asthma phenotypes. Next, we will explore the immunological effect and mechanism of Shp2 regulating DC functions using DCs separating from lungs and bone marrow and DCs treated with lentivirus. The prospective results are helpful to understand the pathogenesis of asthma, and provide the foundation for new drug screening.

环境变应原激发树突状细胞(DC)是哮喘的关键环节，解析DC调控分子机制是哮喘药物研发的新热点。非受体型磷酸酶Shp2是信号调控重要分子，其活化依赖于膜受体锚定结合。Gab1是具有Shp2结合位点的锚定蛋白，也是新发现的哮喘易感因子。前期研究显示Gab1通过参与DC中CCR7信号调控哮喘过程，但其下游磷酸化传导方式尚不明确；预实验结果发现在过敏原作用下，DC中Shp2会被Gab1招募形成复合体并活化，这提示Shp2可作为Gab1下游重要效应元件参与DC功能调控。延续此前工作，本研究构建了Shp2在DC中特异性缺失小鼠(CD11-Cre:Shp2F/F)，首先通过构建敲除小鼠哮喘模型证实了Shp2缺失可显著缓解哮喘；在此基础上通过肺脏分离及骨髓诱导的DC探究Shp2调控DC的免疫学功能；进一步通过原代DC以及慢病毒感染探究其调控机制。预期结果将有助于理解哮喘发病机制，为新药筛选提供依据。

哮喘属于常见的过敏反应，环境和遗传因素共同调控其发病。我们前期的研究发现支架蛋白Gab1可以通过介导CCR7信号通路影响过敏性哮喘的发生，但其下游关键性的磷酸化信号通路尚不清楚。我们的前期研究发现，当外界抗原刺激时，树突状细胞中的Shp2可以被磷酸化活化，这提示我们磷酸酶Shp2可能是哮喘中树突状细胞发挥功能的关键性信号分子。基于此，我们构建了树突状细胞中特异性缺失Shp2的小鼠(Shp2f/fCD11cCre)。在此基础上，我们利用HDM（尘螨）诱导小鼠产生过敏性哮喘。通过分析发现，Shp2缺失小鼠中HDM诱导的过敏性哮喘被缓解。进一步的研究发现Shp2缺失在体内体外都抑制了DC的迁移。机制研究表明，DC中Shp2的缺失增加了p115RhoGEF的磷酸化水平，增强了Rho-GTP的活性。Rho-GTP活性的增加促进了肌球蛋白轻链 (MLC)、cofilin和Pyk2的磷酸化，从而导致DC迁移受损。此外，敲除小鼠Shp2不会影响趋化因子受体7型 (CCR7) 的表达。阻断Shp2对DC抗原的摄取、成熟和呈递没有直接影响。综上所述，我们定义了Shp2通过介导p115RhoGEF/Rho信号来调节DC迁移的新作用，我们的发现了过敏性哮喘一种新的调控机制，对该疾病的治疗具有重要意义。