利用改良全基因组CRISPR扫描技术研究中性粒细胞胞外诱捕网NETs形成机制

Sepsis is a major cause of death in severe trauma and critical ill patients. Since the pathogenesis of sepsis is quite complex, there is no effective prevention and treatment measures. Neutrophils play an important role in launching the first line of host defense against infection. Recently another mechanism-the formation of Neutrophil extracellular traps (NETs) has been discovered, which has been demonstrated to have a close relationship with the morbidity and development of sepsis. However, the molecular mechanism of its formation is still not clear. Genome-wide CRISPR-Cas9 knockout (KO) screens substantially improve the efficiency of loss-of function screens over si/shRNA screens, and our novel computational method further increase the efficiency of CRISPR-Cas9 KO library design to substantially improve screening performance. In order to clarify the formation mechanism of NETs, this project will select out key genes using modified genome-wide CRISPR scanning technology in neutrophil-like cells. Furthermore, the function of those key genes will be analyzed by bioinformatics analysis and a series of in vitro and in vivo experiments. We will also investigate the feasibility of early warning diagnostic methods based on levels of NETs and/or their key genes to predict the morbidity and mortality of sepsis in severe trauma patients. This study aimed to deepen the understanding of the pathogenesis of sepsis, clarify the formation mechanism and role of NETs in sepsis, provide a new target for the treatment of sepsis patients and provide new early-warning diagnostic methods for trauma and critically ill patients as well.

脓毒症是严重创伤和危重症患者院内死亡的主要原因。其发病机制复杂，目前尚无有效防治措施。中性粒细胞胞外诱捕网（NETs）是近年新发现的中性粒细胞杀菌途径，与脓毒症发生、发展及严重程度密切相关，但其形成的具体分子机制尚不明确。课题组前期通过生物信息学分析，对现有CRISPR全基因组扫描技术进行了改良，降低了CRISPR的脱靶效应，为全基因组层面上的功能基因筛选提供了有利工具。本课题拟以类中性粒细胞为模型，采用改良的全基因组CRISPR扫描技术从全基因组层面系统筛选中性粒细胞NETs形成的关键基因，并通过生物信息学分析和系列体内外实验验证，系统阐明NETs形成的具体机制和相关通路，探讨利用其进行脓毒症发生与死亡率预警诊断的临床可行性。研究旨在加深对脓毒症发病机制的认识，阐明NETs形成机制及在脓毒症发病中的作用，为脓毒症患者的治疗提供新靶点, 为创伤和危重症患者提供新的预警诊断方法。

脓毒症是严重创伤和危重症患者院内死亡的主要原因。其发病机制复杂，目前尚无有效防治措施。中性粒细胞胞外诱捕网（NETs）是近年新发现的中性粒细胞杀菌途径，与脓毒症发生、发展及严重程度密切相关，但其形成的具体分子机制尚不明确。我们建立了CRISPR全基因组扫描技术，并发现低切应力可以直接诱导NETs生成。在动脉粥样硬化小鼠模型中，我们发现血管低切应力部位NETs生成明显增多。如果应用NETs生成抑制剂可以明显降低高脂小鼠动脉血管脂质斑块沉积。同时，我们利用转录组测序筛选出了中性粒细胞感应低切应力，生成NETs的关键基因。同时，我们发现了新的创伤并发症早期预警标志物——血浆Vanin-1。通过多中心临床研究，我们发现创伤早期Vanin-1水平与脓毒症、多器官功能衰竭发生率明显相关。在验证人群中，血浆Vanin-1水平预测脓毒症的敏感性和特异性优于PCT、CRP和APARCHE II评分（AUC=0.82,95%CI 0.77-0.87)。Vanin-1还可以提高APACHE II评分的诊断效果(AUC=0.85)。研究结果发表SCI论文5篇，申请发明专利1项，参编专著1部，培养硕士研究生2名，博士研究生1名，并获2019年重庆市产学研创新一等奖。研究加深了对脓毒症发病机制的认识，探讨了NETs形成新机制，并为创伤和危重症患者提供新的预警诊断方法。