利用表面离子共振技术模拟白细胞穿越血管内皮（TEM）过程，阐明CD47调节白细胞TEM机制的研究

Leukocyte transendothelial migration (TEM), which plays an important role in inflammatory immune responses, is regulated by highly complex mechanisms involving numerous factors, such as chemokines, adhesive molecules and cytoskeletal regulators. CD47 (also known as integrin-associated protein; IAP) is a 50-kDa transmembrane glycoprotein universally expressed in almost all cell types, including endothelial cells. CD47 is present on the apical surface and is enriched at endothelial cell-cell junctions. CD47 interacts with integrins, signal regulatory protein (SIRP) alpha, and thrombospondins. But, the mechanisms of CD47-mediated regulation of leukocyte TEM remains poorly understood. In this project, a surface Plasmon resonance (SPR) technique will be used to establish an in vitro system stimulating TEM, which allow leukocytes to slowly flow through living HUVEC cell-coated SPR chip. In order to identify the key signaling pathways downstream of CD47 that regulates leukocyte TEM, we will make the recombinant SIRP? extracellular CD47-binding domain of human, and detect SIRP alpha extracellular domain-induced cytoskeletal rearragenment in endothelial cells. The small G protein Rac takes an essential part of cytoskeleton rearrangement leading to cell contraction, and is activated by CD47 with Gi-sensitive and -insensitive pathway in different cell types. Commonly, Rac is activated by PI3K-Akt pathway under growth factor or by Gi activity under GPCR. Thus, wortmannin, a potent PI3K inhibitor, and PTX, Gi-specific inhibitor, will be used for confirming regulation of CD47 on endothelial in TEM. For further investigation, the dominant negative form of small G proteins will be performed for CD47-induced stress fiber formation in endothelial cells. The proposed studies are expected to not only establish a useful TEM stimulating system, but also provide insight into the mechanisms of CD47-mediate regulation of leukocyte TEM.

白细胞穿越血管内皮细胞（TEM）是炎症反应的一个重要特征之一，参与因子众多而且过程复杂，CD47作为免疫系统中的一个重要因子也参与其中，并通过其对下游信号的多种调控模式，发挥着重要的生理功能。近年来CD47在免疫识别系统、异种移植及糖尿病方面都是一个研究热点，但是对其调控机制的分子水平认识仍然有限。 本次科研旨在利用表面等离子共振技术，通过实时观察白细胞与血管内皮细胞的特异性结合，在体外模拟TEM过程。另外，通过对SIRP alpha细胞外结构域与CD47特异性结合所引起的血管内皮细胞骨架蛋白重排的检测，推测CD47在TEM中对血管内皮细胞的调节机制。并且通过相应的分子生物学手段，确立在TEM过程中CD47对内皮细胞调控所需的关键因子，及其调节机制。为全面了解CD47在体内的生理意义奠定理论基础，同时也为其在临床上的实际应用提供重要依据。

白细胞穿越血管内皮细胞（TEM）是炎症反应的一个重要特征之一，参与因子众多而且过程复杂。CD47是分子量为50 kD的跨膜蛋白受体，通过其配体TSP-1对内皮细胞的功能进行调节。本课题初衷旨通过实时观察白细胞与血管内皮细胞的特异性结合，在体外模拟TEM过程，推测CD47在TEM中对血管内皮细胞的调节机制。.但是在针对CD47缺失小鼠的内皮细胞进行体外分离培养的过程中我们发现，与WT型的内皮细胞相比CD47缺失的内皮细胞具有更持久的连续分裂，更旺盛的增殖以及相对缓慢的细胞衰老等多种现象。针对这一现象我们进行了深入的研究，结果证实了CD47缺失可以促进血管内皮新生，延缓细胞老化，增强内皮细胞体外成管能力，其机制是下调P53和P21表达，以及调节一系列细胞周期关键分子如pRb，CyclinD1，CDK，从而促进内皮细胞增殖和抑制细胞衰老，通过进一步的体内实验发现，CD47缺失同样可以通过下调肿瘤血管内皮细胞中P53和P21表达，加速肿瘤细胞生长并且抑制免疫细胞浸润，坏死面积出现，我们的研究提供了一个通过调节CD47及其下游通路抑制肿瘤血管新生的新的研究方向。并从分子水平解释CD47通过调控细胞周期调节内皮细胞功能以及生存的机制，这也为全面了解CD47在体内的生理意义奠定理论基础，同时也为其在临床上的实际应用提供重要依据。