类风湿性关节炎关中PKCa下调与FOXP3磷酸化和Treg细胞功能降低机制研究

Regulatory T (Treg) cells play a critical role in keeping autoimmunity in check, and impaired Treg cell function is associated with autoimmune pathology of rheumatoid arthritis (RA). In the previous study, we found that FOXP3 phosphorylation enhanced Treg cells function, but FOXP3 phosphorylation and Treg function were decreased in the activated RA patients. Also, the expression level and activity of protein kinase C-alpha (PKCa) reduced in the activated RA patients. However,FOXP3 phosphorylation site, the specific protein kinase and the relationship with Treg function in RA needs to be further investigated. Here we want to study the project from three aspects:① Identification of the FOXP3 phosphorylation site and prepared the specific phosphorylation antibody. ②Identification of the FOXP3 specific protein kinase PKCa and explore the relationship with Treg function. ③ The role of PKCa in the impaird function of Treg cells in RA patients. The innovation of the study is that first to explore the FOXP3 phosphorylation sites and prepare the FOXP3 specific phosphorylated antibody; first to research the role of FOXP3 specific protein kinase PKCa in the function of Treg cells of RA patients. This study will reveal a novel mechanism in which PKCa regulates FOXP3 phosphorylation, thereby altering the Treg function and provide a new theoretical basis and targets for immunotherapty of rheumatoid arthritis.

Treg细胞功能降低是类风关发病的重要机制。本课题组前期研究发现FOXP3磷酸化修饰增强Treg细胞功能，类风关中FOXP3磷酸化水平减少与Treg细胞功能降低有关，类风关中蛋白激酶PKCa表达与活性降低。但FOXP3磷酸化位点，特异性蛋白激酶及其和类风关中Treg细胞功能关系尚不清楚。本课题拟从三方面展开研究:① FOXP3磷酸化位点的确定以及特异性磷酸化抗体制备；②FOXP3磷酸化蛋白激酶PKCa的确立与Treg细胞功能关系。③类风关中PKCa与Treg细胞功能降低的关系。课题创新点在于：①首次探索FOXP3磷酸化位点及制备特异性磷酸化抗体。②首次研究FOXP3磷酸化蛋白激酶PKCa与Treg细胞功能的关系及在类风关中的作用。研究结果将从PKCa和FOXP3磷酸化水平新视点阐明类风关中Treg细胞功能降低的分子机制，有助于拓展类风关发病机制，为免疫治疗提供新的理论基础与靶点。

Treg细胞在抑制自身反应性T细胞增殖，防止自身免疫性疾病的发生中扮演着重要作用，同样Treg细胞数量及功能在类风关中起了重要的调节作用,Treg细胞功能降低是类风关发病的重要机制。本课题组前期研究发现FOXP3蛋白磷酸化修饰能够增强Treg细胞功能，类风关中FOXP3磷酸化水平减少与Treg细胞功能降低有关，类风湿性关节炎中蛋白激酶PKCa表达与活性降低。但FOXP3磷酸化位点，特异性蛋白激酶及其和类风关中Treg细胞功能关系尚不清楚。本课题拟从三方面展开研究:① FOXP3磷酸化位点的确定以及特异性磷酸化抗体制备；②FOXP3磷酸化蛋白激酶PKCa的确立与Treg细胞功能关系。③类风关中PKCa与Treg细胞功能降低的关系。在本研究中我们确立了FOXP3的磷酸化位点，发现FOXP3 S418位点磷酸化能调节FOXP3转录活性和增强Treg细胞抑制功能，并阐明类风湿关节炎TNFa通过促进Treg细胞蛋白磷酸酶1（PP1）的表达和活性，导致Treg细胞FOXP3 S418位点脱磷酸化，从而损伤Treg细胞功能的分子机制，制备了FOXP3磷酸化特异性抗体。另一方面发现了类风湿性关节炎中蛋白激酶PKCa表达与活性降低，但是PKCa并不能明显影响到FOXP3的磷酸化和Treg细胞的功能。研究结果探索FOXP3磷酸化位点S418及制备特异性磷酸化抗体，研究PP1能够特异性下调FOXP3磷酸化水平，并发现蛋白激酶PKCa能够与FOXP3共结合，但是并不能够明显地影响到FOXP3的磷酸化水平和Treg细胞功能。研究结果完善和更新FOXP3蛋白水平决定Treg细胞功能的理论，阐明类风关中Treg细胞功能降低的分子机制，有助于拓展类风关发病机制，为免疫治疗提供新的理论基础与靶点。