基于特征性Tp0136基因序列的神经梅毒诊断方法的建立及其应用

Neurosyphilis has been recognized as a severe disease and difficult to manage, and up to now, laboratory indicator with high specificity for diagnosing neurosyphilis is still unavailable. The enhanced CDC molecular subtyping system is capable of detecting the dominant subtype of Treponema pallidum (Tp) in patients with neurosyphilis. However, employing this method is insufficient to differentiate the neurosyphilis and non-neurosyphilis. The product of Tp0136 is a part of outmembrane protein of Tp, it involves with the adhesion during infection process and it is highly variable in nucleotide sequence. Its variation may lead to the immune evasion, and consequently elevates the risk of developing neurosyphilis. Our pilot study revealed the genetic differences between Tp0136 sequence deprived from neurosyphilis and primary stage syphilis patients, indicating the solid potential for Tp0136 served as molecular biomarker for diagnosing neurosyphilis. The present study attempts to compare the Tp0136 sequence deprived from different types of syphilis patient and different molecular subtypes of Tp, and obtain the specific variations (multiple variations assumed) associated with neurosyphilis by employing bioinformatics; The animal experiment would be conducted to further validate the neuroinvasion caused by the Tp with specific Tp0136 variations, and select five biomarkers for the diagnosis of neurosyphilis. Multi-target real-time PCR system for detecting the specific Tp0136 variations would be established and applied in clinical samples for evaluation. The implementation of present study would improve the early diagnosis of neurosyphilis, and possesses critical value for the prevention and management of syphilis.

神经梅毒的破坏性大、防治困难，缺乏特异的实验室诊断指标。增强分子分型方法可以发现神经梅毒优势亚型，但不足以完全区分神经梅毒和非神经梅毒。Tp0136基因所编码的外膜蛋白在感染过程中参与黏附，且具高度变异性，其变异可能造成免疫逃逸发生神经梅毒；本研究预实验显示神经梅毒和一期梅毒患者Tp0136基因存在异质性，是潜在的神经梅毒诊断指标。本研究拟采用高通量测序比较不同类型梅毒患者、不同分子型别梅毒螺旋体的Tp0136基因序列差异，获得神经梅毒特征性Tp0136变异序列；然后采用生物信息学技术初筛神经梅毒诊断靶标（理论上有多个）；采用动物实验验证具有特征性分子靶标的Tp株的致神经梅毒作用，明确可用于诊断的分子靶标（5个以内）；构建多靶实时荧光定量PCR用于神经梅毒的早期诊断，并开展临床评估。该课题的实施将有助于神经梅毒的早期诊断，对梅毒的防治具有重要的临床价值。

神经梅毒是一种破坏力强、难以防治的疾病，目前尚无特异性的指标用于诊断神经梅毒。有鉴于我国梅毒患病率逐年上升，神经梅毒的患病率也随之增高，因此开展关于神经梅毒诊断以及发病机制的研究具有重要公共卫生意义。本研究通过非靶向代谢组学方法检测了神经梅毒患者、梅毒非神经梅毒患者以及无梅毒感染者的脑脊液，并通过多种统计学方法鉴定出几种具有差异的代谢物，其中部分代谢物在神经梅毒患者脑脊液中显著增高，具有作为诊断标志物的潜质。此外，本研究还通过检测梅毒患者和健康对照的血液代谢物，寻找血液的差异代谢物和进行KEGG分析，鉴定的差异代谢物和代谢途径可为研究梅毒螺旋体的代谢特征提供研究基础。Tp0136是梅毒螺旋体的外膜蛋白之一，其可在梅毒螺旋体对宿主细胞的黏附过程中发挥重要作用。本研究通过将tp0136重组蛋白与HMEC-1细胞共培养，细胞迁移实验显示其迁移活性与tp0136蛋白具有剂量-反应关系，5ug/mL tp0136浓度下细胞覆盖面积为95.7%，与之相似的是，HMEC-1细胞的CCL2 mRNA和蛋白水平也和tp0136蛋白具有剂量-反应关系。通过加入CCL2单克隆抗体和CCR2阻滞剂，发现细胞的迁移活性受到显著抑制。上述结果表明，tp0136蛋白可通过上调CCL2进而促进细胞迁移，其在梅毒螺旋体感染导致的上皮细胞损伤中具有重要作用。

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