原发性干燥综合征LncRNANR_026938对Tfh细胞Unc5cl/ NF-κB通路调控机制研究

Primary Sjögren's syndrome (pSS) patients have abnormal Tfh cell expression and function , and the specific mechanism remains elusive. Previous studies showed significantly higher expression of LncRNA NR_026938 in pSS patients by microarray screening of mRNA and LncRNA, and verified it and its positive correlation with the expression of predicting target gene- Unc5cl ; experiments in vitro further verified that Unc5cl can activate NF-κB pathway, result in Tfh cells expressing CXCR5, ICOS and other important molecular, which assist B cell maturation and the maintenance of ectopic germinal center . Therefore, this project intends to solve the following scientific problems: 1. The mechanisms of LncRNA NR_026938 regulating quantity and function of Tfh cell; 2. The regulatory mechanisms of methylation or transcription factors in the expression of LncRNA NR_026938 in Tfh cells; 3. Using a pSS mice model to explore the possibility of regulationg LncRNA NR_026938 for the treatment and prevention of pSS. Through these studies, it will help us to clarify the regulatory mechanism of LncRNA NR_026938 in immune response of pSS , and provide new targets for pSS therapeutic intervention.

原发性干燥综合征（pSS）患者Tfh细胞表达和功能异常，但其具体作用机制不明。本研究组通过mRNA及LncRNA表达谱芯片筛选并验证发现，pSS患者Tfh细胞中LncRNA NR_026938表达显著增高，且其预测靶基因Unc5cl表达呈正相关；体外实验进一步验证发现，Unc5cl通过NF-κB通路调控Tfh细胞表达CXCR5、ICOS等辅助B细胞成熟和维持异位生发中心形成的重要分子。本项目拟研究以下科学问题：1、LncRNA NR_026938对Tfh细胞数量和功能紊乱的调控机制；2、甲基化或转录因子等对LncRNA NR_026938在Tfh细胞表达的调控机制；3、利用pSS小鼠动物模型，探讨调节LncRNA NR_026938治疗和预防pSS的可能性。通过以上研究，将有助于阐明pSS中LncRNA NR_026938对免疫应答的调控机制，为探讨pSS免疫干预治疗提供新的靶点。

本项目前期研究发现pSS患者Tfh细胞中LncRNA NR_026938表达显著增高，且其预测靶基因Unc5cl表达呈正相关。1. 为了研究LncRNA NR_026938对Tfh细胞免疫调控功能，我们通过病毒转染在Tfh 细胞上实现LncRNA NR_026938功能再现，Imida刺激后ELISA检测发现LncRNA NR_026938过表达组上清液IFN-γ、IL-21显著增高(P<0.05)。将上调LncRNA NR_026938后Tfh 细胞与B 细胞混合，发现IL-21 和IL-10分泌增多，原始B细胞（CD19+CD21+CD27-）百分比下降, 浆细胞（CD19+CD27highCD138+）百分比上调，且IgG、IgA的含量显著增高。2. 为了证明Unc5cl和LncRNA NR_026938存在相互作用。我们分别通过RNA-pulldown技术和RIP实验证明了pSS患者Tfh细胞LncRNA NR_026938和Unc5cl存在相互作用。3. 为了研究pSS患者LncRNA NR_026938表达增高的调节机制。我们通过甲基化特异性PCR(MSP)法检测32例pSS患者和32例健康对照者外周血Tfh 细胞中LncRNA NR_026938启动子区甲基化状态，结果发现pSS患者LncRNA NR_026938启动子区甲基化频率高于健康对照组。我们发现高表达LncRNA NR_026938的pSS患者的Tfh 细胞经DNA甲基转移酶抑制剂5-aza-dc处理后，LncRNA NR_026938启动子区去甲基化，且LncRNA NR_026938的表达显著减低。说明LncRNA NR_026938启动子区的异常高甲基化可能是导致LncRNA NR_026938在SS患者Tfh 细胞中高表达的重要机制。4. 通过SS模型小鼠体内实验发现，过表达LncRNA NR_026938可以通过调节Unc5cl等表达加剧小鼠SS下颌下腺组织淋巴细胞的浸润，进而加速SS病程。本研究初步阐明了LncRNA NR_026938通过调控Tfh 细胞在SS发病机制中发挥作用，为进一步深入理解pSS的发病机理提供了新的思路。