IgG4相关疾病中microRNA对B细胞PI3K-AKT-mTOR通路的调控研究

IgG4 related disease (IgG4-RD) is a newly recognized fibro-inflammatory disease, which is characterized by elevated serum IgG4, swelling and fibrosis of affected organs, accompanied by massive lymphocytes and IgG4 positive plasma cells infiltration of involved organs/tissues. Our previous study revealed that the phenotype and function of peripheral blood B cell subsets were abnormal, as well as the abnormality of immunoglobulin class switch, resulting in large amounts of IgG4 secreting plasmablasts. Our present study is to further investigate the mechanism of B cells activation regulated by microRNA (miRNA) in IgG4-RD. From our previous study, we found the differentially expressed miRNAs were enriched in PI3K-AKT-mTOR signaling pathway compared with Sjogren syndrome and health control. So we will testify 1-2 differentially expressed miRNA in peripheral blood B cells and tissue B cells, and then by knocking down or overexpression of selected miRNA in B cells in vitro, we aim to observe the changes of PI3K-AKT-mTOR signaling pathway activation and B cell phenotype and function. We then will further verify in vivo how the selected miRNA regulating the activation of the PI3K-AKT-mTOR signaling pathway, the mouse model disease phenotype and B cell phenotype and function.

IgG4相关性疾病（IgG4-RD）是新认识的一种以血清IgG4升高、受累器官炎症、硬化，伴大量IgG4阳性浆细胞浸润的疾病。我们前期研究发现IgG4-RD患者外周血中B细胞亚类表达、功能以及免疫球蛋白类别转换异常，导致分泌IgG4的浆母细胞显著升高。本课题将在上述基础上进一步探讨微小核糖体 RNA（miRNA）对IgG4-RD患者B细胞功能的调控。预实验发现，与干燥综合征和健康对照相比，IgG4-RD患者异常表达的miRNA富集于PI3K-AKT-mTOR信号通路。因此我们将验证并确定1-2种差异表达的miRNA，在外周血和组织中B细胞进行相应的敲减及过表达，确证其调节B细胞中PI3K-AKT-mTOR信号通路和B细胞功能的作用；最后，利用IgG4-RD实验鼠模型，体内证实相应的miRNA对PI3K-AKT-mTOR信号通路活化的调控，以及对B细胞活化、分化和疾病表型的影响。

研究目的：探究雷帕霉素靶蛋白(mTOR)通路调控调节性T细胞在IgG4相关性疾病(IgG4-RD)发病机制中的作用。.主要研究内容：收集就诊于北京协和医院的初治和治疗稳定的IgG4-RD患者的外周血，并收集性别年龄相匹配的健康对照(HC)外周血，分离外周血单个核细胞(PMBC)，采用流式方法检测Treg细胞及其TGFβ1和IL-10的表达百分比。收集HC及初治IgG4-RD病人血浆，采用ELISA方法检测血浆中IL-10的表达水平。采用磁珠分选技术，分选出HC和初治IgG4-RD患者的CD4+T细胞，提取RNA逆转录成cDNA、裂解细胞提取蛋白；采取RT-PCR技术检测PTEN、mTOR、TSC1、4EBP1、P70S6K的mRNA表达水平；采用Western Blot技术检测mTOR通路磷酸化PTEN(p-PTEN)、PTEN、p-mTOR、mTOR、p-4EBP1、4EBP1、p-P70S6K、P70S6K的蛋白表达水平。进一步采取流式技术检测HC和IgG4-RD的CD4+T细胞、Treg细胞上的p-PTEN、p-4EBP1表达水平。.重要结果：初治IgG4-RD患者CD4+CD25+Treg、CD4+CD25+Foxp3-Treg的百分比明显高于HC，治疗后均下降；初治IgG4-RD患者CD4+CD25+Treg、CD4+CD25+Foxp3-Treg表面TGFβ1明显高于HC，治疗后明显下降。RT-PCR结果示初治IgG4-RD患者CD4+T细胞的PI3K、PTEN、TSC1在mRNA水平上明显低于HC，治疗后上升，P70S6K、mTOR低于HC，4EBP1治疗后明显上升。Western Blot结果示初治IgG4-RD患者CD4+T细胞中p-PTEN、p-mTOR、p-4EBP1蛋白表达明显高于HC，其余无明显差异。.结论：1.IgG4-RD患者的Treg及其分泌的TGFβ1明显高于HC，治疗后下降，表明Treg分泌的 TGFβ1可能促进纤维化的进程；IgG4-RD患者血浆IL10明显高于HC，并与多项反应病情活动的临床指标呈相关性。2.mTOR通路在IgG4-RD患者CD4+T细胞及Treg细胞高度活化，提示mTOR通路促进Treg细胞增殖，进而参与IgG4-RD发病过程，mTOR通路有望成为IgG4-RD治疗的新靶点。