β2整合素介导的白细胞粘附中Src激酶活化及其力学调控

Cell adhesion, mediated by integrin-ligand interactions, play very important roles in a variety of physiological and pathological conditions such as immune response, coagulation, inflammation, tumor metastasis and wound healing. Integrins on leukocytes play a critical role in their adhesive interactions with endothelial cells. The most unique feature of integrins is their ability to transmit bidirectional transmembrane signaling, which support the dynamic and reversible transformation of integrins between high-medium-low affinity state, and thereby plays a critical role in the regulating cell adhesion. β2 integrin-ICAM-1 are significant effector in adhesion between leukocytes and endothelial cells...The mechanical environment (shear stress) play crucially role in regulating leukocyte adhesion. However, It is unclear where mechano-induced biochemical signals are initiated and how they are transmitted in the cell. Src is well known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli...Here, Using genetically encoded Src biosensor based on Fluorescent Resonance Energy Transfer (FRET), Src activity in HL60 cells and HUVECs mediated by β2 integrin-ICAM-1 interaction are monitored and quantified with high spatial and temporal resolution. And shear stress is introduced to monitor the dynamic process of mechano-transduction induced Src activation. These findings will provide new quantitative understanding of mechano-induced biochemical signals, and could potentially lead to novel therapeutic strategies in the inflammation and immune diseases.

白细胞募集过程可以被认为是一个在流体剪切环境中的粘附和信号分子协同作用的过程。现有研究在外力调控整合素激活、整合素双向信号转导等方面研究取得了重要进展。但是，对于力学因素如何实时动态调控整合素/配体下游信号分子活化以实现其生理功能的研究甚少。本项目拟利用基于荧光共振能量转移的生物探针技术，以白细胞-内皮细胞相互作用为生物学体系，研究在流体剪切作用下β2整合素-ICAM-1介导的白细胞粘附过程中Src激酶的动态分布和活性变化。关键科学问题是白细胞粘附过程中β2整合素-ICAM-1相互作用导致的目标分子的动态变化及力学调控规律，本研究将深化对免疫响应、细胞粘附等重要生理病理机制的认识，为疾病的检测和诊断提供新思路。

白细胞募集过程可以被认为是一个在流体剪切环境中的粘附和信号分子协同作用的过程。本项目以炎症反应过程中白细胞-内皮细胞粘附过程为生物学背景，以力学刺激如何调控整合素-配体介导的白细胞粘附过程的信号转导为科学问题，以基于 FRET 的生物探针为基础、以白细胞/内皮细胞为生物学模型，观察了在流体剪切作用下beta2 integrin-ICAM-1介导的白细胞粘附过程中 Src激酶的动态分布和活性变化，研究了白细胞粘附过程中beta2 integrin-ICAM-1 相互作用导致的目标分子的动态变化及力学调控规律。取得的主要研究成果包括：1）建立了一套能够实现实时测量力学因素调控活细胞内信号分子活化的实验方法和技术手段，建立了测量白细胞和内皮细胞内信号转导的实验方法和分析方法。2）研究了P-和E-选择素对中性粒细胞跨膜迁移动力学的影响，发现P-、E-选择素通过激活beta2整合素介导中性粒细胞跨内皮迁移，其中P-、E-选择素与PSGL-1的结合起主导作用并能激活中性粒细胞较快启动跨膜迁移，而E-选择素与CD44的结合起辅助补充的作用。3）研究了在白细胞（HL60）-内皮细胞相互作用（粘附、爬行、跨膜迁移）中，由粘附分子介导的Src 激酶活化的空间分布和动态变化，发现beta2整合素的不同功能态（中间亲和态和高亲和态）对HUVEC内Src激活有调控作用；E选择素是通过cortactin → Src → myosin II 信号通路来调控内皮细胞胞间链接打开从而促进HL60跨内皮迁移；4）开展了流体剪切调控beta2整合素-ICAM-1 介导的白细胞-内皮细胞相互作用中 Src 激酶活化的实验研究，发现白细胞内Src激活是高亲和性beta2整合素依赖的。本项目研究深化了对免疫响应、细胞粘附等重要生理病理机制的认识。