论文标题:超抗原SEA诱导T细胞失能的分子机制研究
Molecular Cloning and Characterization of Dopamine Responsive Genes From Astrocytes
论文作者 许桂莲
论文导师 朱锡华,论文学位 博士,论文专业 免疫学
论文单位 第三军医大学,点击次数 87,论文页数 135页File Size4652k
2001-05-01论文网 http://www.lw23.com/lunwen_669655167/ 超抗原；SEA；失能；T淋巴细胞；外周免疫耐受；CD28； CTLA-4；负性信号；转录因子；AP-1；NF-kappaB
superantigen; SEA; T lymphocytes; anergy; peripheral immune tolerance; CD28; CTLA-4; negative signaling; transcription factor; AP- 1; NF-kappaB
在免疫学界，免疫耐受一直是研究的重点和难点。失能是机体维持外周免疫耐受的一个主要机制。近年来T细胞失能机制的研究已成为免疫学研究的一个热点。 超抗原是一类新型的抗原分子，它以TCRV_β特异性的方式直接激活大量T细胞。其激活T细胞的独特方式，决定了超抗原具有重要的生物学效应。其中，引起T细胞克隆失能和删除便是其重要生物学影响之一。 为探讨T细胞失能的分子机制，几个研究组进行了一些研究。研究结果表明：失能T细胞中，IL-2基因转录受到伤害是由于TCR活化诱导的p21~(Ras)及其下游MAPK途径受阻所致，即所谓的“Ras-blocked”失能。另外还涉及到转录因子AP-1、NF-κB、NF-AT、c-Fos、FosB、JunB，负性调节因子和RaP1。最近，有研究者在体内诱导的T细胞失能中，发现失能是通过选择性地损害TCR活化诱导的Ca/calcineurin途径来诱导和维持的。少量钙离子载体的加入就能恢复此失能状态，说明这是一种有别于上述失能的“Calcium-blocked”失能。然而，这仅仅是T细胞活化信号的中下游事件。造成这些TCR信号下游受阻事件的上游事件是什么？作为对T细胞活化起关键作用的共刺激分子，CD28/CTLA-4在超抗原诱导的T细胞失能中分别扮演何种角色呢？人们对T细胞失能的胞内生化基础很不清楚。这些问题的阐明对探讨免疫耐受本质的起着关键作用。 超抗原与T细胞相互作用的MHCⅡ类分子非限制性以及大量活化T细胞的特性，使体外失能模型的建立成为可能。本课题通过在体外，用超抗原SEA诱导人外周血T细胞的失能，从而首次建立了超抗原诱导T细胞失能的体外模型，并首次对超抗原诱导的人外周血T细胞失能分子机制进行了研究。实验内容及结果主要包括以下几个方面： 1．超抗原SEA对外周血了细胞的活化特点 初始T细胞接触超抗原后会产生强烈的增殖反应，对抗原的再次刺激，T细胞表现出无反应性。也就是说免疫耐受是T细胞被超抗原活化的后续过程。对超抗原活化T细胞的特点进行了解是研究免疫耐受的前提。 SEA是一种葡萄球菌分泌的外毒素，也是研究最清楚的一种超抗原。我们用超抗原SEA刺激从外周血分离出的PBMC，全面系统地研究了SEA对人外周血 T细胞的活化特点：①采用 3H1dR掺入法实验发现 SEA活化T细胞的最佳浓度范围为 10上0‘＂g／mL。②双抗体夹心 ELISA ＄表明 SEA对T细胞的活化作用在第二、3天达高峰。③通过FACS分析SEA初次刺激和再次刺激的T细胞CD4和CDS表型，发现T细胞不管是初次或再次接触SEA，SEA对CD4“和CDS“T细胞两亚群起同等程度的活化作用，而不改变CD4／CDS表型。 2．建立超抗原SEA诱导了细胞失能的体外模型 研究T细胞失能机制的前提是建立一合适的失能模型。为此，我们做了以下工作：①短期静息SEA反应性T细胞系的建立。首先加入超抗原SEA活化PBMC，72h后，去除SEA，力入rhIL－二（100U／！nL），以扩增SEA反应性T细胞，去除未反应性的细胞。经过两周以上的维持，可得到高纯度的静息SEA反应性T细胞。②证明经过处理后的PBMC可作为超抗原SEA 的抗原提呈细胞。将新鲜分离出的PBMC 经Y．射线照射或用MitoffiyCillC处理后，其增殖能力受到抑制，但保留了其提呈抗原作用。用这种方法处理过的细胞可成功地提呈SEA到T细胞，使T细胞活化。③通过HyPaqueIicoll密度梯度离心法可成功地将经SEA多次刺激过的SEA反应性T细胞与非反应性细胞分离开来，FACS分析所得到的细胞95％以上为SEA反应性T细胞。④证明了在体外经SEA多次刺激过的T细胞处于一无反应状态：将静息短期SEA反应性T细胞与经SEA多次刺激过的T细胞接种培养板，用经上述方法处理过的PBMC作抗原提呈细胞，加 x 入SEA，置于CO。孵箱中培养72h。’H1dR掺入法检测各孔细胞的增殖。 ELISA检测各孔细胞培养48h上清的IL－2浓度。结果表明经SEA多次刺 激过的T细胞处于失能状态，受SEA的再次刺激时，不能增殖，IL上产 生缺乏。从而首次建立了超抗原诱导T细胞失能的体外模型。 3．超抗原SEA诱导丁叁胞失能白分子机未 ①超抗原 SEA诱导的失能 T细胞，IL－ZR a链表达正常，外源性 IL，2 的加入可逆转其失能状态。PMA单独作用能部分恢复T细胞的增殖，但 ＊＊A与1加＊＊yeh的复合作用则更大程度地使T细胞的失能状态得到恢 复。从而首次发现失能T细胞之所以不能增殖，是由于T细胞活化诱导的 信号转导到胞核的Ras／MAPK途径和 Ca／calcineurin途径受阻，从而使IL上 基因不能正常转录。②失能T细胞中，对IL上基因转录起重要作用的转录 因子AP八转录活性减弱，且组份缺陷，这与失能T细胞中IL－2产生缺乏 或减少有关；NF上appaB的转录活性却强于活化组T细胞。其在夫能诱导 和维持中的作用有待进一步探讨。③超抗原SEA诱导的
Molecular mechanism of T lymphocytes anergyinduced by superantigen SEAAbstract Immune tolerance is always of importance in the field of immunology and it is also a problem difficult to resolve. Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In recent years, much attention has been paid to the molecular mechanism of T cells anergy. Superantigen is a kind of novel protein antigens which can directly activate a large number of T lymphocytes in a manner of MHC unrestricted~. TCRV specificity. Its unique activation fasion makes it have special biological effects. Leading to T cells anergy and deletion is one of the important biological effects. In an attempt to understand how anergic T cells maintain their unresponsive state, several groups have analyzed the molecular defects in anergic T cell clones. The results suggest that the impaired transcription of the IL-2 gene in anergic T cells is a consequence of a block in TCR-induced activation of p21 Ras and its downstream mitogen- activated protein kinase(MAPK)pathways. It is also called as “Ras-blocked”anergy. In addition, the involvement of AP-1~ NF-kappaB.~ NF-AT~ c-Fos.~ FosB~. JunB~. negative regulatory factors and Rap 1 has been suggested. In the more recently, a study suggest that in vivo SEB-induced anergic state of T cells is established and maintained by a selective impairment in the TCR-induced activation of Calcalcineurin pathway. It is called as“calcium-blocked”anergy because of theaddition of a small amount of calcium ionophore was able to rescue the anergicstate. However how do the upstream events of TCRsignal transductionpathway affect these blocked downstream evenis? As the critical co-stimulators,CD28/CTLA-4, what roles have they played in the induction and maintenenceof T cel1s anergy by superantigen? All of these questiones remain to explore. Inconclusion, the intracellular biochemical basis underlying the induction ofanergy is stiIl Iargely unknown. To resolve these (" source" questiones is criticalfor the clarification of immune tolerance.Superantigen can activate a large number of T lymphocytes and interactwith T cells in a manner of MHC unstricted, which make the establishmemt ofT cells anergy model induced by superantigen in vitro possible. lt is the firsttime to establish a T cell anergy model and to study the molecular mechanismof T cells anergy ihduced by superantigen SEA in vitro. The experimentalcontents and results are mainly as fOllows:l. Activation of peripheral bIood T cells stimulated by superantigenSEA.Native T cells exposed saperantigen induce a strong proliferative response.After the initial phase of superantigen~induced activation, part of the reactive Tcells are deleted and the remaining T-cell population fails to proliferate andsecrete IL-2 in response to a subsequellt superamigen chal1enge. So in order toinvestigate the mechanism of T cells anergy induced by superaatigen, first ofal1 is to explore the activation of T cells induced by superantigen.StaPhylococcal enterotoxin A is a kind of exotoxins secreted bystaPhyococcus aureus and it is the best-characterized kind of SEs. ln ourexperiments we have investigated the activation properties of PBMC bysuperanigen SEA. OIt is suggested that l0~l04 ng/mL is the optimal SEAconcentration range to activate T cells in vitro by using 3H-TdR incorperationassay. @That the activation arrives at top at the 2. 3 days after the addition ofVISEA analyzed by ELISA. @SEA activates the CD4+ and CD8+T subPopulatioasin the same degree without changing the phenotyPe of CD4/CD8 in the first SEAstirnulation or the restimulation tbrough FACS analySis.2. EstabIi8hment of T cells anergy model induced by superantigenSEA in vitro.We have to establish a suitable T cells anergy model in prior to investigatethe molecular mechanism of T cells anergy. So we have conducted thefOllowing works: OThe short-time resting SEA-reactive T cells l
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