【作者】 卢雪红；

【导师】 李一；

【作者基本信息】 吉林大学 ， 免疫学， 2008， 博士

【摘要】 自身免疫耐受是指免疫系统对自身抗原的特异性免疫无反应状态,是维持免疫自稳的重要机制。自身耐受主要是通过中枢耐受和外周耐受两大机制来维持。为了研究常见的自身免疫性疾病-系统性红斑狼疮(systemic lupus erythematosus,SLE)免疫耐受异常的机制及糖皮质激素对SLE免疫耐受机制的影响,本文首先对SLE患者外周血CD4~+CD25~+调节性T细胞(Treg)进行研究,并分别应用慢性移植物抗宿主病(cGVHD)诱导的SLE小鼠和自发的MRL/lpr狼疮小鼠,全面系统地探讨了自身免疫耐受机制异常在SLE发生中的作用。一、SLE患者外周免疫耐受机制异常为了探讨Treg和Foxp3与SLE发病的相关性,检测了SLE合并狼疮性肾炎患者经肾上腺糖皮质激素(简称激素)冲击治疗后外周血单个核细胞(PBMC)中Treg和相关基因Foxp3表达的变化。结果表明激素冲击治疗后的SLE患者Treg/CD4~+T比值高于正常对照组;Foxp3 mRNA水平表达与对照组差异不显著。Treg数量和功能的变化提示外周免疫耐受机制异常参与SLE患者的发病,激素可能通过提升Treg纠正外周免疫异常而对SLE起治疗作用。二、cGVHD诱导SLE模型免疫耐受机制异常和激素的治疗作用首先采用cGVHD诱导SLE狼疮模型,检测了该模型的发病相关指标、细胞和体液免疫功能。在此基础上,本实验采用激素治疗SLE小鼠模型,以探讨cGVHD诱导的SLE狼疮模型中枢和外周免疫耐受异常的机制。结果表明:1、SLE模型鼠中枢和外周免疫耐受机制异常参与其发病:胸腺异位基因唾液蛋白2(saliva protein 2,SP2)和组织蛋白酶L (cathepsin L,CTSL)的表达明显减弱,与SLE模型鼠的唾液腺炎及全身结缔组织病明显相关;胸腺和脾脏CD4~+CD25~+Treg/CD4~+T比值无明显降低,Foxp3明显减弱,但CTLA-4和GITR表达增加。说明Treg功能减弱而T细胞活化增强参与cGVHD诱导的SLE狼疮模型的发病。2、激素治疗后可部分恢复SLE模型鼠的免疫耐受异常:胸腺中SP2和CTSL表达有一定程度的恢复;胸腺和脾脏中CD4~+CD25~+Treg/CD4~+T无明显变化,Foxp3表达增强,但CTLA-4和GITR表达水平减弱。说明治疗后Treg功能增强而T细胞活化减弱,激素通过纠正中枢和外周免疫耐受的异常而发挥治疗作用。三、MRL/lpr鼠免疫耐受机制异常的研究为了了解MRL/lpr小鼠免疫耐受的缺陷,我们检测了10周龄MRL/lpr小鼠的相关症状,对其中枢和外周免疫耐受异常的机制进行了研究。结果表明:1、10周龄MRL/lpr小鼠,表现出和人类SLE相似的症状。2、MRL/lpr小鼠存在中枢耐受缺陷:胸腺多种异位基因,包括SP-2、CTSL、甲状腺球蛋白(TG)、C-反应蛋白(CRP)的表达水平明显下降,与SLE模型鼠的唾液腺炎、甲状腺炎及全身结缔组织病相关。胸腺CD4~+CD25~+Treg/CD4~+T数量和功能基因Foxp3与对照组无明显差别,而CTLA-4表达增强。3、MRL/lpr小鼠存在外周耐受的异常:脾脏淋巴细胞增殖缺陷,Th1/Th2失衡;CD4~+CD25~+Treg/CD4~+T无明显减少,模型组与对照组相比,Foxp3表达减少,CTLA-4表达增强,提示CD4~+CD25~+Treg在外周的功能缺陷参与其发病。综上, SLE患者存在外周免疫耐受异常,不论cGVHD诱导的SLE模型,还是MRL/lpr小鼠,都存在着中枢耐受和外周耐受机制的异常。基于SLE患者和动物模型的发病机制研究,将为人类SLE提供有效的预防和治疗方案奠定基础。更多还原

【Abstract】 Autoimmune disease (AD) is a kind of disease caused by harmful immune responses between auto antigens and potentially pathogenic auto reactive antigens, which result in tissues and/or organs destruction and loss of physical functions. Self-tolerance is controlled by central and peripheral mechanisms. To investigate the disorder of central and peripheral tolerance in Systemic lupus erythematosus (SLE) and effect of glucocorticoid therapy on immunotolerance, three experiments were designed and completed to clarify the issue.Part 1 Change of CD4~+CD25~+T cell in SLE patientsIn order to investigate the effect of CD4~+CD25~+Treg cell and its functional gene (Foxp3) on SLE, CD4~+CD25~+Treg cell and Foxp3 were detected in peripheral blood mononuclear cells (PBMC) of SLE patients after treatment by glucocorticoid. The results showed that the number of CD4~+CD25~+Treg was significantly enhanced in PBMC from SLE patients. The expressions of Foxp3 mRNA were not significantly changed. It means that CD4~+CD25~+Treg may be responsible for pathogenesis of SLE and glucocorticoid may increment CD4~+CD25~+Treg frequency to treat SLE patients.Part 2 The abnormities of central and peripheral tolerance in SLE modelIn order to clarify the pathogenesis of SLE, cGVHD induced SLE murine model was made and role of promiscuous gene expression in thymus and CD4~+CD25~+ regulatory T cell were investigated on this model. 1. The establishment of SLE model(Balb/c×C57BL/6) F1 mice were made by 6-8 weeks-old Balb/c and C57BL/6 mice. With the injection of lymphocyte separated from spleens, thymus and lymph nodes of Balb/c mice to F1 mice, the SLE models were established successfully by the method of chronic graft versus host disease (cGVHD). 4 week after the last injection, the model group got the phenomena of loosing hairs, particularly in snouts, and activity reduced. Urine protein in the model group was positive (~+~+~~+~+~+), but in the control group were negative. Anti-nuclear antibody (ANA) in serum was positive for all the model mouses. There were no positive results in the control and the negative control group. All kidney slices of the model group showed various degrees of green fluorescence. This result demonstrated that the immunocomplex deposited in glomcrulus and most were IgG..Glomerulus’s hyperplasia, thickening basement and lots of inflammatory cells infiltration were found in the kidney slice of the model group mice. And in the mean time, inflammatory cells were infiltrated in salivary glands.2. Role of promiscuous gene expression in thymus and CD4~+CD25~+ regulatory T cell on immunotolerance of SLE modelThe thymic promiscuous gene expression of CTSL and SP2 declined in the model group. Obvious sialadenitis and general symptoms in SLE model mice may be relevant with this phenomenon. The quantities of CD4~+CD25~+Treg and the ratio of Treg/CD4~+T were not significantly changed both in thymus and spleen. Foxp3 declined, but CTLA-4 and GITR increased in model group. The results indicated that both Treg function decreased and T cell activated contributed to the pathogenesis of SLE model.3. Effects of glucocorticoid therapy on the central and peripheral tolerance for SLE modelIn order to clarify the role of the glucocorticoid therapy on central and peripheral immunotolerance, model mice were divided into control and therapy group randomly. The therapy group was injected with glucocorticoid. The model group was injected with normal saline (NS). Less trichomadesis phenomenon was found in therapy group. New hairs were found in trichomadesis mice of the therapy group. However, the model group mice did not improve. Urine protein of the model group were ~+~+~~+~+~+, and the counterpart of the therapy group mice were ~+~~+~+. The fluorescence intensity in the therapy group mice was less obviously than that in the model group. The symptoms in therapy group were improved greatly by glucocorticoid. The promiscuous gene expression of CTSL and SP2 in the therapy group was higher than that in the model group. The thymic promiscuous gene expression has recovered by the therapy of glucocorticoid. Quantities of CD4~+CD25~+Treg were not changed after the therapy, but the expression of Foxp3 recovered and CTLA-4 and GITR decreased in the therapy group. The results showed that the function of Treg reinforced obviously and T cell activation was depressed by glucocorticoid.Part 3 The abnormities of central and peripheral tolerance in MRL/lpr miceMRL/lpr mice showed that Anti-nuclear antibody was positive in model group but negative in control group. The thymic promiscuous gene expression of CTSL, CRP, TG and SP2 declined in MRL/lpr mice. There were no significant differences of quantities of CD4~+CD25~+Treg in thymus for model and control group.Foxp3 levels of model group were no significant difference in thymus but lower in spleen compared with that of control group. CTLA-4 levels of model group were higher than control group both in thymus and in spleen. These result suggest that MRL/lpr mice does not occur as a result of reduced level of CD4~+CD25~+Treg, but function defect of CD4~+CD25~+Treg may be responsible for its occurrence.更多还原