【作者】 佟海滨；

【导师】 曾宪录；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2013， 博士

【摘要】 中性粒细胞(Polymorphonuclear neutrophils，PMN)是人体外周血中含量最多的白细胞，是参与早期炎症反应的重要免疫细胞之一，迁移对于中性粒细胞发挥其生理功能有着重要的意义。当机体遭受外来细菌或其他病原体入侵时，中性粒细胞在第一时间被募集到感染部位，充当了机体的第一道防线，这种防御机制是建立在中性粒细胞迁移和趋化基础上的。在各种趋化物的作用下，中性粒细胞凭借黏附受体以及胞内分子对炎性信号的优化和放大，能够迅速极化并准确向炎症部位形成定向迁移，发挥免疫清除作用。中性粒细胞是一把“双刃剑”，在某些病理条件下导致的中性粒细胞的大量组织浸润，可以引发过度炎症反应，常常造成机体组织的严重伤害，是急性肺损伤、急性呼吸窘迫综合征等炎症相关疾病的主要成因。因而，深入揭示中性粒细胞迁移的分子机制、筛选干预中性粒细胞迁移的作用靶点，将具有重要的生理及病理意义。中性粒细胞迁移依赖多种黏附分子的参与，其中β2整联蛋白对于中性粒细胞与活化血管内皮间的稳定黏附以及迁移起着重要作用。β2整联蛋白家族是白细胞特异性表达的黏附分子，它们由一个共同的β亚基分别和四种α亚基构成以非共价键结合的异源二聚体。静息状态下，β2整联蛋白呈现低亲和力构型。中性粒细胞在选择素介导的滚动黏附作用以及趋化因子的刺激下，β2整联蛋白能够迅速活化并与其配体结合，通过整联蛋白的“outside-in”信号调节细胞骨架变化，从而影响细胞稳定黏附、铺展及迁移。但是，对于β2整联蛋白介导的中性粒细胞迁移的分子调控机制尚不明确。在中性粒细胞中，β2整联蛋白与生理配体的交联刺激能够活化胞内多种酪氨酸激酶，通过激活这些酪氨酸激酶向胞内传递骨架调节信号，影响细胞骨架重塑及细胞迁移。c-Abl激酶是一种重要的非受体酪氨酸激酶，具有广泛的生物学效应，它主要受到黏附受体以及生长因子受体的调控，参与黏附受体以及生长因子受体引起的信号转导事件，调控包括细胞周期、细胞凋亡、基因表达、DNA损伤修复以及F-actin依赖的细胞骨架变化在内的一系列重要细胞活动进程。有文献报道，在多种细胞类型中，整联蛋白家族成员均可以活化c-Abl激酶，并且参与调控细胞的稳定黏附与铺展。本文将主要探讨c-Abl非受体酪氨酸激酶在β2整联蛋白介导的中性粒细胞迁移过程中的生理功能及作用机制。本论文综合运用急性腹腔炎小鼠模型、Transwell及琼脂糖悬滴迁移模型，从体内、外层面评价c-AbI激酶对中性粒细胞迁移的影响，利用活细胞显微成像分析系统实时观察并记录c-AbI激酶活性对中性粒细胞迁移行为的影响，为揭示c-AbI激酶可能的作用环节提供线索。采用流式细胞术、免疫荧光，GST pull-down,免疫共沉淀、体外激酶反应等实验技术与方法深入揭示β2整联蛋白介导的中性粒细胞迁移进程中c-AbI激酶的可能作用机制。通过本论文的研究发现，c-AbI激酶活性在中性粒细胞向炎症部位的募集以及β2整联蛋白介导的中性粒细胞迁移过程中起着重要的作用。当c-AbI激酶活性受到抑制后，中性粒细胞的组织浸润和迁移行为受到严重影响，同时，中性粒细胞迁移前缘的形成及细胞膜的外向伸展受到显著抑制。当fMLP刺激激发细胞建立新的黏附联系时，导致β2整联蛋白与配体充分交联，这一过程能够触发c-AbI激酶活化，并在β2整联蛋白介导的F-actin细胞骨架重塑事件中发挥重要作用。Rho家族小G蛋白(Rho Small GTPase)作为“分子开关”，参与调节中性粒细胞骨架的动态变化，是调控中性粒细胞黏附、极化、迁移的核心分子。Rho GTPase家族成员在鸟苷酸交换因子(Guanine-nucleotide exchange factor, GEF)的催化下，由GDP结合的非活化状态向GTP结合的活化状态转换。Rac1是Rho GTPase家族中的重要成员，它通过参与调节骨架动态与片层伪足形成影响细胞极化、黏附与迁移。Vav1作为活化Rac1的主要GEF，可以在黏附受体的下游调节肌动蛋白细胞骨架重排，参与了β2整联蛋白依赖的中性粒细胞铺展与稳定黏附。我们的工作发现，在β2整联蛋白介导的中性粒细胞迁移过程中，c-AbI激酶与Vav1存在共定位现象，并利用免疫共沉淀技术证实二者存在于同一功能复合物中，通过GST pull-down分析发现c-AbI激酶与Vav1二者是直接结合的， Vav1中C-末端的SH3、SH2结构域和脯氨酸富集区是介导Vav1与c-AbI激酶相互作用的关键结构域。通过体外激酶实验发现Vav1是c-AbI激酶下游重要效应分子，c-AbI激酶活性影响β2整联蛋白依赖的Vav1磷酸化水平。利用Scansite磷酸化位点预测分析以及定点突变技术，发现Vav1的Dbl同源（DH）结构域中Tyr-267是c-AbI激酶的主要磷酸化位点。c-AbI激酶对中性粒细胞迁移的调节作用，可能是通过影响Vav1/Rac1介导的细胞骨架重塑而实现的。更多还原

【Abstract】 PMN is an essential component of the innate immune system. When acute inflammationoccurs, neutrophils are rapidly recruited and extravasated from blood into infection sites,where they destroy the invading microorganisms by phagocytosis of pathogens and releasingseveral antimicrobial chemicals. However, the excessive and improper recruitment ofneutrophils usually result in serious tissue injury. Therefore, understanding the molecularmechanism underlying the recruitment and migration of neutrophil is of great physiologicaland pathological importance.Cell migration is a highly complicated and regulated process, in which the regulation ofactin cytoskeleton plays a pivotal role by promoting the formation of membrane protrusions atthe leading edge and providing a driving force, together with molecular motors, to move thecell body. Molecules involved in regulating actin cytoskeleton rearrangement have been wellidentified. A variety of adhesion molecules is required for neutrophil migration, in which theβ2integrin plays an important role in the stable adhesion between neutrophils and activatedvascular endothelial and transendothelial migration. β2integrin family is highly expressedadhesion molecules in the neutrophil cell surface, they are formed by a common β subunitwith four α subunit, forming non-covalent heterodimer. In resting neutrophils, β2integrin ispresent in low-affinity state. Once the stimulation of chemokines, β2integrin is rapidactivated, and adjust the dynamic changes of the cytoskeleton by the"outside-in" signalingpathway, and ultimately regulate cell migration movement. However, the molecularmechanism for the β2integrin-mediated neutrophil migration is still unclear. The engagementof β2integrin can activate a variety of non-receptor tyrosine kinase, induce cell cytoskeletonrearrangement. c-Abl kinase, a nonreceptor tyrosine kinase expressed ubiquitously inmammalian cells, serves as an important link in signal transduction pathways that promoteactin dynamics and cytoskeletal rearrangement. Especially, c-Abl kinase is a key factorinvolved in the formation of membrane ruffling and cell spreading, as well as the dynamicformation or extension of filopodia and lamellipodia in response to extracellular signal or adhesion stimulation. However, the mechanisms underlying the regulation of actincytoskeleton by c-AbI kinase and the downstream effector of c-AbI kinase, as well as theirroles in neutrophil migration, have not been well identified.The rearrangement of actin cytoskeleton is highly influenced by the activity of Rho familyGTPases. One extensively studied family of GEF, which is responsible for the enzymaticactivity of Rho GTPases, is the Dbl family proto-oncogene Vav. Vav1, the first discoveredmember, is a hematopoietic, cell-specific signal protein, and it is involved in the alterations ofcell shape and motility by triggering cytoskeletal changes, as demonstrated by its ability toinduce membrane ruffles and lamellipodia formation.In the present study, a signaling mechanism that controls actin polymerization andmembrane protrusion dynamics at the leading edge during β2integrin-dependent neutrophilmigration is well-defined, with c-AbI kinase playing a central, regulatory role. In addition, wefind Vav1colocalizes and interacts with c-AbI kinase at the leading edges of migratingneutrophils. Vav1is phosphorylated directly by c-AbI kinase at Tyr-267in the DH domain.Taken together, we demonstrate a critical role of c-AbI kinase in neutrophil migration byregulating Vav1activity, providing the feasibility to target c-AbI kinase for the amelioration ofacute inflammation-related diseases.更多还原