体液免疫在神经梅毒中枢神经系统中启动、维持和调控机制的研究

发布时间：2018-06-12 11:46

本文选题：梅毒螺旋体 + 神经梅毒　；参考：《安徽医科大学》2017年博士论文

【摘要】：第一部分 CXCL13/CXCR5在神经梅毒中枢神经系统中对B细胞的招募作用研究背景:梅毒是由梅毒螺旋体感染引起的一种性传播疾病,神经梅毒可以发生在梅毒螺旋体感染机体后的始终。越来越多的证据表明免疫应答参与了神经梅毒中枢神经损伤,其中体液免疫发挥了重要的作用。而迄今为止神经梅毒患者中枢神经系统中体液免疫的启动和维持机制尚不明确。研究方法:使用流式细胞术分析111个梅毒患者脑脊液样本中的B细胞及B细胞亚群的分布情况。样本包括23个无症状神经梅毒、43个有症状神经梅毒和45个非神经梅毒,其中分别有36个和17个随访病人符合随访要求。使用ELISA法检测脑脊液中免疫球蛋白(IgM,IgA和IgG)、CXCL13的浓度。使用改良后的24孔小室进行趋化实验和中和实验,分析不同组别梅毒患者的脑脊液对B细胞的趋化作用;CXCL13中和抗体抑制神经梅毒患者脑脊液样本中的CXCL13后,分析趋化后B细胞比例和B细胞亚群的变化情况。使用免疫组化法检测鞘内梅毒瘤病变组织的中spirochete、CXCR5、CD20+B细胞、CD3+T细胞、CD138+浆细胞,和CD35+滤泡状树突状细胞及其分泌的趋化因子CXCL13的表达情况。研究结果:我们发现:相较于非神经梅毒,神经梅毒患者脑脊液中B细胞的比例显著升高(P0.001),并且B细胞处于激活状态。在未治疗的神经梅毒患者免疫球蛋白指数(IgM,IgA和IgG指数)显著高于非神经梅毒,并且与疾病的进展有相关性。神经梅毒患者脑脊液中的CXCL13的表达水平显著升高(P0.001)。此外,脑脊液中高水平的CXCL13可以在体内和体外介导B细胞向中枢的迁移。更重要的是,神经梅毒患者脑脊液中的B细胞、免疫球蛋白指数以及CXCL13的水平三者之间均呈显著的正相关(P值均0.05)。最后,神经梅毒患者脑脊液对于B细胞的趋化指数显著高于非神经梅毒;脑脊液预先使用CXCL13中和抗体孵育后,可以阻止其对B细胞的趋化作用。脑实质梅毒瘤病变组织中含有滤泡样结构,其包含大量的B细胞、T细胞和浆细胞,以及在淋巴细胞间网状排列的滤泡状树突状细胞,并且表达CXCL13。研究结论:神经梅毒患者中枢神经系统中CXCL13/CXCR5可以介导外周血B细胞的聚集,通过形成异位生发中心促进体液免疫应答。这些结果为阐明神经梅毒的中枢神经损伤机制以及免疫治疗提供了新的线索。第二部分 Th1/Th2相关细胞因子对IgG亚类在神经梅毒患者脑脊液中分布的调控研究研究背景:神经梅毒中枢神经系统中存在激活的体液免疫应答,在局部产生大量的免疫球蛋白,不仅有利于梅毒螺旋体在局部的清除作用,同时也可能参与中枢神经系统的损伤作用。由于免疫球蛋白的产生以及抗体型别和类别转换依赖细胞因子的微环境,而IgG不同的亚类的功能也有所不同,所以我们检测神经梅毒患者脑脊液中IgG亚类的分布情况以及细胞因子对其的调控作用。研究方法:采用超敏多因子电化学发光技术检测126例脑脊液样本中的固有免疫细胞因子(IL-1β,IL-6,IL-8,TNF-α),Th1细胞相关的细胞因子(IFN-γ,IL-2,IL-12p70)和Th2细胞相关的细胞因子(IL-4,IL-10,IL-13);这126例样本包括15个正常对照,无症状神经梅毒23例,有症状神经梅毒43例,非神经梅毒45例。其中符合第一次随访和第二次随访要求的神经梅毒病人各有31例和15例。使用ELISA法检测脑脊液样本中IgG1,IgG2,IgG3和IgG4的浓度。研究结果:我们发现相较于非神经梅毒和正常对照组,神经梅毒患者脑脊液中的固有免疫细胞因子(IL-1β,IL-6,IL-8,TNF-α),Th1细胞相关的细胞因子(IFN-γ,IL-2,IL-12p70)和Th2细胞相关的细胞因子(IL-4,IL-10,IL-13)均显著高于正常对照(P0.05)和非神经梅毒(P0.05)。神经梅毒患者脑脊液中IgG1,IgG2,IgG3和IgG4的浓度显著高于非神经梅毒(P0.05)和正常对照组(P0.001),然而非神经梅毒和正常对照组之间IgG1,IgG2,IgG3和IgG4的浓度无显著差异。神经梅毒患者中枢神经系统中Th1和Th2呈现协同共存作用,分别分泌IFN-γ和IL-13,并且这两种细胞因子与免疫球蛋白亚类(IgG1,IgG2,IgG3,IgG4)呈正相关,特别是IgG1和IgG3。在随访中发现,神经梅毒患者脑脊液中IgG1,IgG2和IgG3在第一次治疗后(P0.05)显著下降,然而在第二次随访中IgG3的水平较治疗前无显著差异(P=0.147)。研究结论:神经梅毒患者脑脊液中的IFN-γ和IL-13参与脑脊液中IgG亚类的调控,主要是IgG1和IgG3的类别转换;IgG3,作为一个保护性免疫球蛋白,可能可以作为评估神经梅毒患者临床治疗效果评判指标。
[Abstract]:The first part of the study on the role of CXCL13/CXCR5 in the recruitment of B cells in the central nervous system of neurosyphilis: syphilis is a sexually transmitted disease caused by Treponema pallidum infection. Neurosyphilis can occur after the infection of the body of Treponema pallidum. More and more evidence suggests that the immune response is involved in the neurosyphilis central God. After injury, humoral immunity plays an important role. And to date, the mechanism of starting and maintaining humoral immunity in the central nervous system of neurosyphilis is not clear. Methods of flow cytometry were used to analyze the distribution of B cells and B subsets in the cerebrospinal fluid samples of 111 syphilis patients. The samples included 23 asymptomatic samples. Neurosyphilis, 43 symptomatic neurosyphilis and 45 non neurosyphilis, of which 36 and 17 patients were followed up for follow-up. The ELISA assay was used to detect the concentration of immunoglobulin (IgM, IgA and IgG) and CXCL13 in the cerebrospinal fluid. The modified 24 pore chamber was used for chemotaxis experiment and neutralization test to analyze different groups of syphilis patients. The chemotactic effect of cerebrospinal fluid on B cells; CXCL13 neutralization antibody inhibited CXCL13 in cerebrospinal fluid samples of patients with neurosyphilis, analyzed the proportion of B cells after chemotaxis and the changes of B cell subsets. Immunohistochemistry was used to detect spirochete, CXCR5, CD20+B cells, CD3+T cells, CD138+ plasma cells, and CD35+ in the tissue of intrathecal syphilitic lesions. The expression of follicular dendritic cells and their secreted chemokine CXCL13. Results: we found that compared to non neurosyphilis, the proportion of B cells in the cerebrospinal fluid of patients with neurosyphilis increased significantly (P0.001), and the B cells were in the active state. The immunoglobulin index (IgM, IgA and IgG index) in patients with untreated syphilis of the syphilis (IgM, IgA and IgG) It is significantly higher than non neurosyphilis and is associated with the progression of the disease. The level of CXCL13 expression in the cerebrospinal fluid of patients with neurosyphilis increased significantly (P0.001). In addition, the high level of CXCL13 in cerebrospinal fluid can mediate the migration of B cells to the center in vivo and in vitro. More importantly, the B cells in the cerebrospinal fluid of the neurosyphilis patients, the immune ball. There was a significant positive correlation between the protein index and the level of the level of CXCL13 (P value is 0.05). Finally, the chemotactic index of cerebrospinal fluid to B cells in patients with neurosyphilis was significantly higher than that of non neurosyphilis; cerebrospinal fluid was incubated with CXCL13 neutralization antibody in advance, which could prevent the chemotaxis of B cells from the B cells. A follicular structure contains a large number of B cells, T cells and plasma cells, as well as follicular dendritic cells in the reticular arrangement of lymphocytes, and the expression of CXCL13. studies: CXCL13/CXCR5 can mediate the aggregation of peripheral blood B cells in the central nervous system of neurosyphilis patients and promote humoral immunity through the formation of ectopic germinal center. These results provide new clues to elucidate the mechanism of neurosyphilis's central nervous injury and immunotherapy. Second the study of the regulation of the distribution of the IgG subclass in the cerebrospinal fluid of patients with neurosyphilis by Th1/Th2 related cytokines: the activation of the humoral immune response in the neurosyphilis central nervous system, in the Bureau The ministry produces a large number of immunoglobulin, which is not only conducive to the local scavenging of Treponema pallidum, but also may be involved in the damage of the central nervous system. Because of the production of immunoglobulin and the conversion of antibody type and category to the microenvironment of cell factors, the function of the subclass of IgG is different, so we examine the function of the subclass. The distribution of IgG subclass in cerebrospinal fluid of patients with neurosyphilis and the regulation of cytokines in the cerebrospinal fluid. Methods: using hypersensitivity multifactor electrochemiluminescence technique to detect the innate immune cell factors (IL-1 beta, IL-6, IL-8, TNF- alpha) in 126 samples of cerebrospinal fluid and Th1 fine cell related cytokines (IFN- gamma, IL-2, IL-12p70) and Th2 cells Related cytokines (IL-4, IL-10, IL-13); these 126 samples included 15 normal controls, 23 asymptomatic neurosyphilis, 43 symptomatic neurosyphilis, and 45 non neurosyphilis, of which 31 and 15 of the patients met the first and second follow-up requirements of neurosyphilis. IgG1, IgG2, IgG3 in cerebrospinal fluid samples were detected by ELISA. And IgG4 concentration. Results: we found that the innate immune cell factors (IL-1 beta, IL-6, IL-8, TNF- alpha) in cerebrospinal fluid of patients with neurosyphilis compared to non neurosyphilis and normal control group, and Th1 cell related cytokines (IFN- gamma, IL-2, IL-12p70) and Th2 cell related cytokines (IL-4, IL-10,) were significantly higher than those of normal controls. P0.05) and non neurosyphilis (P0.05). The concentration of IgG1, IgG2, IgG3 and IgG4 in the cerebrospinal fluid of patients with neurosyphilis was significantly higher than that of non neurosyphilis (P0.05) and normal control group (P0.001). However, there was no significant difference in the concentration of IgG1, IgG2, IgG3 and IgG4 between the non neurosyphilis and the normal control group. IFN- gamma and IL-13 were secreted by CO coexistence, and the two cytokines were positively correlated with the subclass of immunoglobulin (IgG1, IgG2, IgG3, IgG4), especially in IgG1 and IgG3. in follow-up. The IgG1, IgG2, and IgG3 in the cerebrospinal fluid of patients with neurosyphilis decreased significantly after the first treatment (P0.05), however, at the second follow up levels No significant difference before treatment (P=0.147). Conclusions: IFN- gamma and IL-13 in cerebrospinal fluid of patients with neurosyphilis participate in the regulation of IgG subclass in cerebrospinal fluid, mainly the category conversion of IgG1 and IgG3; IgG3, as a protective immunoglobulin, may be used as a marker for evaluating the clinical therapeutic effect of patients with syphilis.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R759.13

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