亲神经亚型梅毒螺旋体在神经梅毒发生中的作用机制

The pathological mechanism of neurosyphilis is unknown. Many researches have focused on host immune response, however, overlooking the genetic background of Treponema pallidum (Tp). Studies have shown that some genotypes Tp may be related to the pathogenesis of neurosyphilis. Our preliminary studies found that Tp from cerebrospinal fluid of patient with neurosyphilis was more resistant to the removal of phagocytic cells than Tp from chancre lesions of patient with acute syphilis. We guess that there should be some neurotropic Tp subtypes and a quite number of neurosyphilis are caused by them. This project attempts to proceed with Tp genotyping, then find the related information about China's neurotropic Tp subtypes. And the differences in genes and proteins will be detected by using the full gene sequence analysis and proteomic analysis. Secondly, making research on the activation, proliferation, apoptosis and changes of related cytokine levels of immune cells mediated by the crucially different recombinant Tp protein. So as to we can explore the role of this recombinant Tp protein in immune imbalance and suppression of cellular immunity. Animal model experiment will be further performed to study the changes of host immune function and outcome mediated by neurotropic Tp subtypes and its key differences proteins in vivo, revealing how the neurotropic Tp subtypes cause immunologic derangement and escape immune clearance of host. We research on the present difficult and blind zone for the pathogenesis and injury mechanism of neurosyphilis from a new perspective. It is expected to make a major contribution on the breakthrough of TP study.

神经梅毒发病机制未明，其研究集中在宿主的免疫应答反应上，而忽视梅毒螺旋体（Tp）遗传背景。研究表明某些基因型别Tp可能与神经梅毒发病有关，我们前期研究也发现神经梅毒患者脑脊液来源的Tp比急性梅毒患者硬下疳来源的Tp更能抵抗吞噬细胞的清除。推测存在亲神经亚型Tp，并且相当数量神经梅毒的发生由其介导。本项目从Tp基因分型入手，寻找我国亲神经亚型Tp信息，采用全基因序列分析和蛋白质组学技术发现其差异基因和蛋白质；研究亲神经Tp关键蛋白介导免疫细胞活化、增殖和凋亡，促进巨噬细胞吞噬作用，诱导免疫细胞表达前炎症细胞因子，探索关键蛋白在细胞免疫失衡和免疫抑制中的作用；并通过动物模型，研究该亚型Tp及其关键蛋白介导宿主免疫功能变化和结局，揭示亲神经亚型Tp导致机体免疫功能紊乱，逃逸免疫清除，发展为神经梅毒的作用机制。从全新角度阐明神经梅毒的发病和损伤机制等当前神经梅毒研究的难点和盲区，有望产生重要突破。

研究背景：未经治疗，或治疗不彻底的梅毒患者中有 25 %-40% 将发展为神经梅毒。至今对梅毒螺旋体感染中枢神经系统后，发生自然消退和产生严重神经系统性损伤的机制一无所知。研究表明某些基因型别梅毒螺旋体可能与神经梅毒发病有关。.研究方法：我们采用CDC subtype/Tp0548 sequencetype 分型系统，检测神经梅毒的分型，并与非神经梅毒比较，寻找亲神经亚型梅毒螺旋体信息。采用蛋白质组学技术筛查差异蛋白质。进一步研究差异蛋白在神经梅毒免疫中的作用。.研究结果：我们采用63例临床可疑神经梅毒患者的脑脊液接种新西兰兔睾丸，神经梅毒脑脊液的接种阳性率为2.78%（1/36），非神经梅毒脑脊液的接种阳性率为3.70%(1/27)，获得2株梅毒野生株基因型分别为14d/f 和14a/f。采用密度梯度离心法收集梅毒螺旋体，提取膜蛋白进行差异蛋白分析。应用所获得的差异蛋白进行体外细胞实验，发现神经梅毒差异蛋白能抑制巨噬细胞的吞噬作用，导致螺旋体逃逸了巨噬细胞的吞噬。而后续的动物实验也发现神经梅毒组的外周血淋巴细胞的CD3+CD8+ 淋巴细胞的高于对照组(χ2 =4.427, P = 0.035)，而NK细胞的低于对照组。（χ2 =13.226, P = 0.000），存在免疫抑制。.研究结论：存在神经梅毒基因亚型，神经梅毒基因亚型和非神经梅毒基因亚型在体外免疫吞噬和对机体的免疫功能的影响不同，为后续神经梅毒免疫抑制机制的研究提供了实验依据。