树突状细胞来源外泌体miRNAs作为梅毒血清固定诊断标志物的筛选及其应用

The incidence of syphilis serofast is high and there is lack of early diagnostic markers. Aiming to look for the key molecules closely associated with the serofast as diagnostic targets is the orientation of diagnostic research. Our preliminary findings revealed that Treponema Pallidum (T.pallidum) had a subtype which had an advantage on causing serofast reaction. The exosomes of dendritic cells (DCs) induced by the subtype T.pallidum mediated the imbalance Th1/Th2 and the polarization of macrophage to M2 type. Consequently, the occurrence of T.pallidum immune evasion developed the serofast. Moreover, the miRNAs presented specific in the exosomes of DCs derived from the serofast patients. It is speculated that the characteristic miRNAs in exosomes would be the potential early diagnostic markers of the serofast. Aiming to verity the hypothesis we proposed：first, the different expression level of miRNAs in the exosomes from DCs induced by the serofast subtype T.pallidum would be screened by high-throughput sequencing on the HiSeq-2500, Illumina, second, the optimal combination of miRNAs would be determined by decision tree analysis to build syphilis serofast diagnostic model and then the specific miRNAs would be validated by the clinical (theoretically there are multiple miRNAs), finally, the universal fluorescent tagged miRNAs microarray would be constructed to exclusively detect the specific miRNAs for early diagnosis of the serofast and the clinical performance would be evaluated. The study is expected to establish early diagnostic model of the serofast to resolve this long-term clinical problem, and it has an important clinical value in the syphilis serofast prevention and treatment.

梅毒血清固定现象发生率高，缺乏早期诊断指标。寻找与其发生密切相关的关键分子作为诊断靶标，是诊断研究的方向。我们发现：存在致梅毒血清固定优势亚型梅毒螺旋体（Tp），其作用于树突状细胞（DC）后分泌的外泌体介导Th1/Th2细胞失衡，巨噬细胞向M2极化，发生Tp免疫逃逸，引发血清固定；该类患者DC来源外泌体存在特征性miRNAs。推测外泌体特征性miRNAs是潜在的血清固定早期诊断指标。本项目拟开展：1)采用Illumina HiSeq-2500高通量测序平台，初筛致血清固定亚型Tp感染DC分泌的外泌体差异表达miRNA；2）应用决策树分析法选出最佳miRNA组合，建立梅毒血清固定诊断模型，临床验证确定特征性miRNA（理论上有多个）；3）构建通用荧光标签miRNA芯片检测特征性miRNA，用于血清固定早期诊断，并开展临床评估。本研究有望解决梅毒血清固定早期诊断这一难题，对梅毒防治有重要意义。

近年来，血清固定一直是我国乃至世界范围的重要社会公共健康问题，尽管其无临床表现，临床意义也存在争议，但是部分患者却具有传染性，同时会发展成三期梅毒，给梅毒的有效防治造成了巨大困难。项目分析梅毒螺旋体作用后树突状细胞的外泌体miRNA，共获得55个差异miRNA，采用聚类分析进一步筛选梅毒血清固定的差异miRNA，发现梅毒螺旋体作用后miR-122-5、miR-133b和miR-278-3a明显上调，miR-16、miR-341-5p和miR-500明显下调。采用血清固定临床样本对所筛选的差异miRNA进行验证，梅毒血清固定的miR-122-5、miR-133b和miR-278-3a的明显高于非血清固定组，两组miR-16、miR-341-5p和miR-500没有差异。构建的miR-122-5、miR-133b和miR-278-3a芯片技术用于差异miRNA的检测，并对构建的miRNA芯片检测技术进行临床应用研究，miRNA芯片检测技术诊断梅毒血清固定的灵敏度为80.00%，特异性为82.00%。为梅毒血清固定诊断提供新的指标。与此同时该项目扩展性的对血清固定患者来源外泌体中的蛋白进行了检测，发现了血清固定患者来源外泌体中存在Tp蛋白，为后续梅毒致病机制的深入研究提供了新方向。该项目培养中青年骨干2名，培养研究生3名，共发表SCI论文13篇，较好地完成了课题任务。