长链非编码RNA-ENST00000421645调控Th1细胞参与神经梅毒发生的机制

Up to date, the pathogenesis of neurosyphilis remains unclear. Our preliminary experiment indicated that neurotropism subtype Treponema pallidum (T.pallidum) was closely associated with the occurrence of neurosyphilis, showing the former probably induces the imbalance of Th1/Th2 cells to develop neurosyphilis. Furthermore, the expression of lncRNA-ENST00000421645 in CD4+ T cells was significantly elevated in peripheral blood samples acquired from patients with the infection of neurotropism subtype T. pallidum. Hypothesis that when neurotropism subtype T.pallidum infects the organism, T. pallidum would up-regulate the expression of lncRNA-ENST00000421645 and consequently the function of Th1 cell would be suppressed. Through above mentioned process, this specific subtype of T. pallidum involves in the pathogenesis of neurosyphilis. Aiming to verity the hypothesis we proposed: first, the expression of lncRNA-ENST00000421645 in Th cells of neurotropism subtype T. pallidum infected patients would be determined. Second, in vitro experiment would be conducted to investigate the molecular mechanism of the up-regulation of lncRNA-ENST00000421645 and the suppression of the Th1 cell function caused by the stimulation of neurotropism subtype T. pallidum. Moreover, the immune evasion of T. pallidum caused by the IFN-γ secretion and the compromised phagocytosis of microglial cells after the suppression of the Th1 cell function would be extensively investigated. Finally, animal model with the infection of neurotropism subtype T. pallidum would be employed to verify the pathogenesis of neurosyphilis mediated by suppressed Th1 cells function caused by the differential expressed lncRNA-ENST00000421645. The present study attempted to probe in the pathogenesis of neurosyphilis with a novel insight, and expected to identify the potential biomarker for the diagnosis of neurosyphilis.

神经梅毒发病机制未明。我们发现亲神经亚型梅毒螺旋体（Tp）与神经梅毒的发生密切相关，前者可能通过介导Th1/Th2细胞的免疫失衡，导致神经梅毒发生；且亲神经亚型Tp感染的患者外周血Th细胞中lncRNA-ENST00000421645显著升高。推测亲神经亚型Tp感染机体后，通过上调该lncRNA而抑制Th1功能，参与神经梅毒的发生。为验证假设，首先明确亲神经亚型Tp感染的神经梅毒患者Th细胞中该lncRNA含量；其次，采用体外实验探讨亲神经亚型Tp刺激Th1表达该lncRNA，抑制Th1功能及其分子机制；并阐明该lncRNA抑制Th1功能后，分泌IFN-γ影响小胶质细胞吞噬功能，引发Tp免疫逃逸的作用；最后经动物模型验证亲神经亚型Tp感染机体后，该lncRNA抑制Th1细胞活性介导神经梅毒发生的作用过程。本研究从全新的角度阐明神经梅毒发病机制，并为神经梅毒的鉴别诊断寻找新靶标。

梅毒螺旋体导致神经系统损伤致病未明。近期研究发现，某些长链非编码RNA（lncRNA）参与调控机体免疫应答，以应对病原体的入侵。神经梅毒患者外周血T淋巴细胞中lncRNA表达与非神经梅毒患者存在显著差异。本课题进一步阐明lncRNA-ENST00000421645在神经梅毒发病机制中的作用。. 本项目明确神经梅毒患者CD4+T细胞中 lncRNA-ENST00000421645含量最高，显著高于非神经梅毒患者。对经梅毒患者、非神经梅毒患者CD4+T细胞中 lncRNA-ENST00000421645 结果进行ROC曲线分析结果，曲线下面积为0.730，P=0.011，有统计学意义；说明lncRNA-ENST00000421645对神经梅毒的鉴别诊断有意义。细胞定位实验结果显示，lncRNA-ENST00000421645主要位于Jurkat细胞的胞浆中。与慢病毒空载体（NC细胞）相比，LncRNA-ENST00000421645过表达慢病毒转染的Jurkat细胞（OE细胞）在PMA刺激下促进T淋巴细胞分泌IFN-γ。RIP 测定证明 lncRNA-ENST00000421645 与PCM-1蛋白相互作用。沉默PCM1显著增加PMA刺激的OE细胞中IFN-γ的表达。根据lncRNA表达谱分析，与lncRNA-ENST00000421645相邻的基因为Kank1。通过Western印迹分析表明OE细胞中Kank1的表达显著上调。实验鉴定lncRNA-ENST00000421645作用于乙酰化酶NAT10。染色质免疫沉淀（ChIP）-PCR结果显示与NC细胞相比，OE细胞组蛋白H3K27组中Kank1基因启动子的富集显著增加3.96倍，表明lncRNA-ENST00000421645促进了Kank1启动子附近组蛋白H3K27的乙酰化，从而促进了Kank1蛋白的表达。实验进一步表明Kank1促进14-3-3蛋白表达，抑制NF-kB活化，抑制T淋巴细胞分泌IFN-γ，促进T淋巴细胞凋亡。