【作者】 陈继冰；

【导师】 李一；

【作者基本信息】 吉林大学 ， 免疫学， 2007， 博士

【摘要】 自身免疫耐受是机体的免疫系统在生理状态下对自身组织表现的“免疫不应答”现象。这种不应答状态是由中枢耐受和外周耐受双重机制来完成的。其中胸腺在诱导T细胞的中枢耐受过程中起重要作用。大部分自身反应性T细胞在胸腺通过阴性选择启动细胞程序化死亡而被克隆清除。胸腺基质细胞表达的多种组织限制性抗原(tissue-restricted antigen,TRA)是参与对自身反应性T细胞进行阴性选择的重要分子。这种生理状态下胸腺基质细胞表达多种TRA基因的现象被称为“异位基因表达”。胸腺“异位基因表达”对T细胞进行着阴性选择。与自身肽-MHC分子复合体具有高度亲和力的T细胞(自身反应性T细胞克隆)特异性识别自身抗原肽后将发生凋亡而被清除。自身免疫调节因子(Autoimmune regulator,Aire)主要表达在胸腺髓质上皮细胞(medullary thymic epithelial cell,mTEC),该基因的突变与人类多腺体的自身免疫病发生有关,并且Aire基因敲除小鼠的部分胸腺“异位基因表达”降低。因此推测这一基因在正常生理状态下通过调节“异位基因表达”维持着自身免疫耐受。但目前缺乏Aire调节胸腺异位基因表达的直接证据。本课题对Aire在“异位基因表达”中的调节作用进行了如下研究:1、分离并培养出较高纯度的Balb/c鼠胸腺上皮细胞,研究了mTEC中Aire及多种TRA表达情况。发现mTEC中Aire及多种TRA表达随培养时间的延长而逐渐减弱。2、通过转染Aire真核表达质粒,发现Aire可促进体外培养mTEC中TRA表达,并延长其表达时间;胸腺细胞的存在可进一步促进Aire转染mTEC中TRA的表达;Aire可通过促进mTEC中TRA表达促进自身反应性T细胞凋亡。3、通过对两种1型糖尿病动物模型(STZ诱导模型和NOD鼠)的研究,发现1型糖尿病动物胸腺中多种TRA表达水平明显降低,CD4+CD25+调节性T细胞(Regulatory T cells, Treg)产生数量减少。4、通过转染Aire,可恢复NOD鼠mTEC中缺陷的多种TRA表达,且胸腺细胞可增强Aire对缺陷的多种TRA的恢复作用。本研究为揭示中枢免疫耐受机制提供实验依据,为人工操纵免疫耐受探索新的途径。免疫耐受机制的揭示和研究具有重要的生物学意义和临床应用价值:通过诱导移植物、变应原以及自身抗原耐受,防治移植排斥反应、I型超敏反应和自身免疫病;通过打破肿瘤和病原微生物的耐受,激发有效肿瘤免疫应答和提高抗感染免疫。更多还原

【Abstract】 Self-tolerance is the non-response of immune system to self components in physiological conditions. Two kinds of mechanisms are involved in maintaining self-tolerance: central tolerance and peripheral tolerance. Thymus plays important roles in central tolerance. Various immature T clones that express particular TCR could be produced by random rearrangement of TCRαand TCRβgene segments during the maturation of thymus. The immature T clones that have high affinity with tissue restricted antigens (TRA) are induced to apoptosis and deleted. The deletion of self-reactive T clones in thymus is called negative selection. Several thousands of TRAs could be expressed by mTECs and DCs in cortico-medullary junction, which is called promiscuous gene expression. Promiscuous gene expression of thymus might play an important role in negative selection of self-reactive T clones. However, the mechanisms that could be involved in regulation of promiscuous gene expression are not well understood.Autoimmune regulator (Aire), whose mutation is associated with the disease of autoimmune polygland syndrom 1 (APS I) in human, is mainly expressed in mTEC. The expression of many TRAs in thymus decrease in Aire knockout mice, So Aire might play the function in negative selection by regulating the promiscuous gene expression in physiological conditions.In this research, we have studied the regulatory functions of Aire on promiscuous gene expression from two parts: Part I. Effects of Aire on TRA expression in mTEC of Balb/c mice1. Purity and identification of primarily cultured TECThe primarily cultured TECs were identified by immunochemistry methods. The purity of Balb/c-TEC increases with time and at d8 the purity could be about 85%.2. Aire and TRAs expression in primarily cultured mTECThe expression level of Aire and TRAs in mTEC was analyzed By RT-PCR. The result showed that the expression level of Aire and TRAs in Balb/c-mTEC attenuated gradually as culture time, was higher at d3, weaker at d8 and disappeared at d11.3. Effects of Aire on TRAs expression of Balb/c-mTECAire was transfected transiently to mTEC at different culture days-before and after the disappearance of promiscuous gene expression-and expression of TRAs was detected by RT-PCR. The results showed that Aire could promote TRAs expression only at being transfected before the disappearance of TRA expression, and different TRAs were regulated at different levels by Aire. Aire could extend the expression time of TRAs in mTEC. Thymocytes were important resource of upstream activation signals of Aire.4. Effects of Aire on inducing thymocytes to apoptosis by mTECIncreased TRAs expression in mTEC by transfected with Aire could promote apoptosis of T cells.Conclusions:a. The expression level of Aire and TRAs in Balb/c-mTEC attenuated gradually with culture time.b. Aire can promote the expression level and extend the expression time of TRAs in mTEC at the early phase of culture. Thymocytes are important resource of activation signals of Aire protein.c. The ability of Aire-mTEC in inducing apoptosis of thymocytes improved obviously.Part II. Effects of Aire on TRAs expression in mTEC of T1D models1. TRAs expression and Treg production in thymus of STZ-induced models There were obvious“Three highs and one low”signs of diabetes mellitus, the urine glucose were all strong positive, serum IAA levels increased obviously with apparent islitis in experimental group, which showed that T1D models were induced by STZ successfully. The thymi of model mice diminished much and lost normal physiological structure of cortico-medullary junction. In the mean time, the whole CD4+CD25+Treg populations decreased which might be an important reason of T1D.Expression of TRAs in thymus of experimental group decreased obviously, which might be important for T1D because of abnormal central tolerance.2. TRAs expression and Treg production in thymus of NOD miceThe body weights of NOD mice we bought(10-12 week old,female) were similar with Balb/c mice essentially, the urine glucose were all negative, the blood glucose levels were all normal, simultaneously with the volumes of thymi. On the other hand, serum IAA levels of NOD mice were higher than Balb/c mice and many pathobiologic changes took place in several glands (e.g.,infiltration of inflammatory cells in islets、salivary glands、thyroids and mixing of cortico-medullary junction in thymus). Production of CD4+CD25+Treg decreased and the decreased part might be the antigen-specific Treg, not the whole Treg library. MHCII molecules expression on NOD-mTECs was lower than Balb/c-mTEC obviously, which might be an important reason of disorder in negative selection of NOD mice.The expression of Aire and TRAs in NOD-mTEC decreased obviously, which showed manifest defects in promiscuous gene expression of NOD-mTEC. The expression of Aire and TRAs in NOD-mTEC attenuated gradully as culture went on, similar with Balb/c-mTEC.3. Effects of Aire on TRA expression in NOD-mTECAire could recover the defected expression of TRAs in NOD-mTEC and extend the expression time. The thymocytes can promote the recovery of Aire to defected TRAs expression in NOD-mTEC.Conclusions:a. There are several defects in thymus of T1D models, which might be important reasons of T1D.b. Aire can recover the defected TRAs expression of NOD-mTEC, and thymocytes can promote the recovery of Aire to defected TRAs expression in NOD-mTEC.Innovation of this thesisa. Transfering-cultured mTEC cell lines lost TRA expression during the long-time culture procedure and can’t be used in research of promiscuous gene expression. We established an Aire transiently-transfecting model of primarily cultured Balb/c-mTEC initiately,and depending on this model we researched the effects of Aire on promiscuous gene expression of thymus and found direct proofs of Aire to promote the TRA expression in mTEC.b. On the basis of the discovery that Aire can promote promiscuous gene expression of thymus, we researched the cross-talk between thymocytes and stromal cells in thymic microenviroment. We found that thymocytes might promote the regulation of Aire to TRAs expression in mTEC, Aire can promote apoptosis of thymocytes by promoting TRA expression in mTEC. c. After discovering the defects of TRAs expression in thymi of T1D models, we then established another model of Aire transiently-transfecting NOD-mTEC. We found that Aire can recover the TRA expression in pathological mTEC.Position and value of the resultsThe research methods in this thesis have never been found in or out of our country, and the results might bring fresh methods and outlets to the research of promiscuous gene expression in mTEC. Maybe they can play important roles in the investigation of acting mechanisms of Aire, TRA expression in mTEC and the etiological factors of autoimmune diseases, meanwhile bring significant evidences to the gene therapies of multiple autoimmune diseases.更多还原