【作者】 巴雪青；

【导师】 曾宪录；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2004， 博士

【摘要】 血流状态下白细胞的募集是一个非常快速的过程，需要特别的机制建立白细胞与内皮细胞间的联系，选择素家族代表了一类适应这一特殊目的的黏附分子。选择素及其配体可在血流状态下介导白细胞在内皮细胞上的起始黏附。起始黏附不仅引发了白细胞向炎症部位的募集，此外，淋巴细胞的归巢与再循环，造血祖细胞向骨髓中归巢、血栓形成、动脉粥状硬化的形成、损伤修复以及肿瘤细胞的转移等生理或病理过程的关键也依赖于循环状态细胞在血管内皮细胞上起始黏附的这一附着过程。 选择素是近年来发现的一类黏附分子，是血管系统内表达的Ca²⁺依赖的动物凝集素，包括P-selectin（选择素P，CD62P）、E-selectin（选择素E，CD62E，）和L-selectin（选择素L，CD62L，）三个成员。象所有的动物凝集素一样，唾液酸化的路易糖X（SLe^x）或它的同分异构体（SLe^a）是三种选择素都选择性识别的主要配体结构。另外，三种选择素都对某些带有SLe^x或SLe^a寡糖侧链的大分子（糖蛋白或糖脂）表现出较高的亲和性。 PSGL-1（P-selectin glycoprotein ligand 1，P选择素糖蛋白配体1）是一种组成性地表达于几乎所有白细胞微绒毛上的黏蛋白。许多in vivo和in vitro实验证明，三种选择素与PSGL-1都有较高亲和力。而且，新近的研究表明，PSGL-1不仅起到从血流中捕获白细胞的作用，作为一种信号分子，它引发了白细胞活化过程中一系列的胞内信号事件，包括β₂-整合素活化、蛋白酪氨酸磷酸化、细胞因子的分泌及基因转录的活化等。 在白细胞活化过程中，F-actin细胞骨架的动态变化扮演了重要的角色。细胞形态变化依赖细胞骨架的变化，白细胞在归巢或炎症反应过程中需要细胞形态发生剧烈的改变，以适应其在内皮细胞上附着、铺展及外移。已知许多白细胞黏附分子如LFA-1、CD28、CD31及L-selectin等起始的信号可引起F-actin细胞骨架的变化，那么，起始黏附过程中PSGL-1在与配体结合时，转导的胞外信号是否也可引起白细胞F-actin细胞骨架的改变?为此，我们分离制备了人外周血嗜中性粒细胞，以其为实验材料，对膜上的PSGL-1分子进行抗体交联，实验结果显示，PSGL-1抗体交联引起嗜中性粒细胞活化后，PSGL一1与细胞骨架结合，成为一种去垢剂不溶的成分，而且，细胞内的F一actin产生了聚合和重新分布，伴随F一actin为结构基础的这种细胞骨架的变化，PSGL一1发生极化，重新分布于细胞F一actin集中的一端。细胞松弛素B的处理，消除了PSGL一1的这种极化现象。上述实验结果提示，PSGL一1转导的信号可活化嗜中性粒细胞，诱导F一actin骨架发生聚合和重新分布，并在F一actin骨架的指导下产生PSGL一1分子的极化。 除了细胞质中F一actin细胞骨架发生改变是本实验观察到的一个由PSGL一1介导的信号事件外，我们通过瞬时转染报告基因质粒、荧光素酶活性分析及RT一PCR技术，进一步在基因转录水平检测到PSGL一1转导的胞外信号可引起Jurkat细胞核内基因CSF一1转录的上调。 近年来，非受体酪氨酸激酶在白细胞的信号通路中的重要作用受到广泛关注。本文研究工作中所提及的PSGL一1介导的白细胞信号中，是否也存在酪氨酸激酶的参与?我们着重探讨了c一Abl和Syk分别在上述两种信号事件中的作用。 我们在实验中观察道，PSGL一1抗体交联引起中性粒细胞活化后，。一Abl重新分布并定位于F一actin集中的细胞周边或突起处，而且，c一Abl酪氨酸激酶特异的抑制剂STI571抑制了活化白细胞中F一actin的聚合和重新分布，实验结果提示，PSGL一1抗体交联促使中性粒细胞活化过程中，c一Abl参与了由PSGL一1到F一actu为结构基础的细胞骨架的改变这一信号通路。 此外，共转染实验结果显示，过表达Syk显著促进了抗体交联PSGL一1引发的Jurkat细胞中CsF一1转录活性的上调。而过表达Syk激酶区突变的共转染实验，及syk特异性抑制剂Piceat^ol对抗体交联后内源CSF一1的mRNA表达的抑制实验进一步说明了Syk的酪氨酸激酶活性参与了抗体交联PSGL一1引起的 CSF一1基因转录活性上调这一信号通路。 综上所述，本文首次报道了PSGL一1介导的信号对F一actin为结构基础的细胞骨架的影响。并首次揭示了酪氨酸激酶c一Abl在这一过程中所起到的重要作用。另外，本文工作确认了一种新的受PSGL一1信号调控的靶基因csF一1，并探讨了酪氨酸激酶syk在这一信号传递通路中的参与。本文实验结果作为新的证据进一步证实了PSGL一l是一种信号受体分子，可转导胞外信号并引发白细胞内酪氨酸激酶参与的的信号事件。关键词:PSGL一l，信号转导，F一aetin细胞骨架，CSF一l，e一Abl，Syk乙毕尹更多还原

【Abstract】 The recruitment of leukocytes from the flowing bloodstream is a very rapid process that requires special mechanisms for the establishment of leukocyte-endothelium contacts. The selectins represent a class of cell adhesion molecules that is specialized for this purpose. Selectins and their ligands mediate the capture of leukocytes from the bloodstream and rolling of leukocytes along the endothelial cell surface. This initial contact is named rolling adhesion, which initiates not only the recruitment of leukocytes to inflamed tissue but also the homing and recycling of lymphocytes, the homing of hemopoietic progenitor to the marrow, thrombopoiesis, atherosclerosis, wound healing and tumor metastasis as well.Selectins, a new-found family of adhesion molecules, are Ca2+-dependent zoo-agglutinins expressed within vascular system and consist of P-, L- and E-selectin. Like all zoo-agglutinins, the tetrasaccharide sialyl Lewis* and its isomer sialyl Lewis are binding determinants for three selectins. Three selectins all show their high affinity to some macromolecules carrying sialyl-Lewis oligosaccharides (glycolipid or glycoprotein).PSGL-1 (P-selectin glycoprotein ligand 1) is a mucin expressed on microvilli of almost all types of leukocytes. Accumulating in vivo and in vitro studies have shown that PSGL-1 is a high-affinity ligand of three selectins. In addition to its direct role in capture of leukocytes from the bloodstream, PSGL-1 also has been demonstrated to function as a signal-transducing receptor and initiate a series of intracellular signal events during the activation of leukocytes, including activation of P2-integrin, tyrosine-protein phosphorylation, secretion of cytokines and transcriptional activation as well.The dynamics of F-actin-based cytoskeleton plays a important role in the process of leukocytes activation. Cytoskeleton dynamics is the basis of cell morphological changes, and leukocytes require drastic morphological changesduring homing and inflammation to enable themselves adhere and spread on the endothelial cells and transmigrate out of the endothelial barrier. Signals initiated from many leukocyte adhesion molecules (LFA-1, CD28, CD31 and L-selectin) can induce the alteration of the F-actin-based cytoskeleton. In order to investigate if the engagement of PSGL-1 and its ligand can also induce the alteration of the F-actin-based cytoskeleton in leukocytes during rolling adhesion, we isolated and prepared human peripheral blood neutrophils and crosslinked PSGL-1 with mAb. The results showed that PSGL-1 associated with cytoskeleton and became a detergent-insoluble component after antibody crosslinking. In addition, F-actin filaments assembled and redistributed, and along with this alteration of F-actin, PSGL-1 polarized to the end of cell where F-actin located. Treatment of cells with cytochalasin B eliminated the polarization of PSGL-1. Taken together, the results suggested that signals transduced by PSGL-1 can activate neutrophils and induce the assembly and redistribution of F-actin, and the polarization of PSGL-1 is under the direction of F-actin-based cytoskeleton.Except for the alteration of F-actin, we detected a new transcriptional-level signal event by the technique of transient transfection, luciferase assay and RT-PCR, that is the engagement of PSGL-1 promoted the transcriptional activity of gene CSF-1.Recently, the participation of non-receptor tyrosine kinase within the leukocyte signal pathway is an area of intense research. To determinate if there are non-receptor tyrosine kinase involved in the signal events observed in our study (the alteration of F-actin and the transcriptional activation of CSF-1), we emphatically investigated the role of non-receptor tyrosine kinase c-Abl and Syk respectively in the two signal pathways.First, we showed that c-Abl was redistributed and re-localized to the periphery or an end of the neutrophil where F-actin concentrated after antibody crosslinking of PSGL-1. Furthermore, STI571, a special inhibitor for c-Abl, obviously blocked the alteration of F-acti更多还原