P-选择素糖蛋白配体1（PSGL-1）介导中性粒细胞募集在新生儿脓毒症中的作用及机制研究

Neonates, in contrast to adults, are featured with the defect in their innate immunity-associated antimicrobial responses, which renders them more susceptible to bacterial infection and sepsis. Our previous work has shown that neonates display inefficient innate immune functions, characterized by the impaired polymorphonuclear neutrophil (PMN) recruitment and antimicrobial activity. We further revealed that neonatal PMNs in comparison with adult PMNs exhibited significantly less P-selectin glycoprotein ligand 1 (PSGL-1), leading to diminished PMN chemotaxis and recruitment. Blockage of PSGL-1 in adult PMNs also caused reduced PMN recruitment; however, the precise molecular mechanism involved is still unclear. In this proposed research project, we will use clinical samples and in vitro experiments to investigate the influence of neonatal PSGL-1 expression and distribution on PMN recruitment and PMN-associated bactericidal activity. We will also use the PSGL-1 neutralizing antibody and PSGL-1 knockout mice to clarify the underlying mechanism responsible for PSGL-1-modulated PMN recruitment and antimicrobial activity. Finally, we will use the in vivo animal model to verify the correlation between the defective expression of PSGL-1 and the high vulnerability of neonates to microbial sepsis. By identifying the significance of PSGL-1 in neonatal innate immunity against microbial infection and sepsis, this study will help us to clarify PSGL-1 as a new immuno-therapeutic target for prevention and treatment of microbial sepsis in neonates.

由于新生儿赖以抵抗微生物感染的固有免疫反应存在缺陷,导致新生儿病患对感染引起的脓毒症易感性升高。我们前期研究发现：新生儿中性粒细胞（PMN）募集能力低下、抗菌作用不足,与新生儿固有免疫反应缺陷密切相关，进而研究发现新生儿PMN P-选择素糖蛋白配体1（PSGL-1）表达低下，影响PMN的募集能力，并且干预PSGL-1亦导致PMN募集被削弱，但确切机制尚待研究。本项目拟利用临床标本及动物模型进一步分析新生儿PSGL-1的表达与分布对PMN募集与抗菌作用的影响；并通过中和抗体与基因敲除小鼠对PSGL-1干预，分析PSGL-1影响中性粒细胞募集与抗菌作用的机制；同时采用动物体内实验探讨PSGL-1表达缺陷与新生小鼠对病原微生物高易感的确切分子机制。本项目通过阐明PSGL-1干预新生儿脓毒症的作用机制，从而为新生儿脓毒症的免疫干预提供一种新的靶点。

新生儿和婴幼儿的感染，包括细菌性脓毒症，是人类面临的非常重要的健康问题。我们对于新生儿免疫反应的认识和研究远远落后于成人。由于新生儿的适应性免疫系统发育尚不成熟，使得其主要依靠固有免疫，而中性粒细胞快速而有效的募集至感染部位是抵抗微生物感染的关键步骤。在本项目中，我们以新生鼠和成年小鼠建立脓毒症小鼠模型为研究对象，①通过比较各组模型动物的存活率，证实新生小鼠比成年小鼠对脓毒症感染的易感性明显增高；②检测小鼠腹腔灌洗液中的PMN数量以及血液、重要器官中的载菌量，明确新生小鼠向感染部位募集的PMN influx明显削弱，清除细菌能力降低；③分离新生儿脐血和成人外周血中PMN，发现PSGL-1低表达是新生儿PMN募集与杀菌功能降低的内在机制；④进一步体外细胞实验在PMN中拮抗PSGL-1表达，发现血小板-PMN复合体的形成、PMN的迁移、吞菌、杀菌功能被抑制；⑤再次新生小鼠在体实验评估确定PSGL-1表达缺陷与新生小鼠对脓毒症感染高易感的相关性。本项目研究结果揭示了PSGL-1表达缺陷是新生儿PMN募集与杀菌功能降低的内在机制，为新生儿脓毒症的免疫干预和防治提供一种新的靶点。