VIM基因低频变异在新生儿脓毒症中的易感性分析和作用机制研究

新生儿脓毒症（NS）发病率和死亡率高，发病机制不明，诊疗方案有限且疗效欠佳。VIM基因在NS中表达升高，且Vim敲除小鼠显示伤口愈合困难；但VIM在NS中的作用及分子机制不明。我们前期发现NS患儿富集VIM低频变异，且VIM可能通过SYK基因在NS中发挥作用。本课题拟：在中性粒细胞带荧光斑马鱼中用CRISPR/Cas9和单细胞显微注射技术构建VIM野生/变异型斑马鱼模型，脂多糖诱导脓毒症发生后明确VIM变异在NS中的易感性；利用免疫共沉淀和双荧光素酶报告基因实验明确VIM对SYK的调控；慢病毒转染小鼠骨髓中性粒细胞过表达VIM野生型/突变型，脂多糖诱导后CCK8、流式细胞术和Transwell检测变异对细胞增殖、凋亡、吞噬和趋化的影响，RNA测序、WB和ELISA探索VIM-SYK/信号通路的调控网络，阐明VIM变异在NS中的作用机制。项目将为NS的早期识别和潜在药物靶标提供新的理论依据。

Neonatal sepsis (NS) has high incidence, mortality and poor prognosis in neonatal population. The pathogenesis of NS is unclear, and the methods of clinical diagnosis and treatment are limited and have poor effect. The expression level of VIM gene is increased in newborns with sepsis. Meanwhile, Vim knockout mice have defects in wound healing and tissue repairing after injury. However, currently the role and molecular mechanism of VIM gene in NS are unclear. We previously found that low frequency variants of VIM gene were enriched in newborns with sepsis and VIM gene might play a role in NS through SYK gene. We plan to carry out this project: ⑴to construct zebrafish model with VIM variants by CRISPR/Cas9 and single-cell microinjection methods on Tg(mpx:EGFP) zebrafish, which has green fluorescent protein in neutrophils, and to make clear the susceptibility of VIM variants in NS after adding lipopolysaccharide (LPS); ⑵to clarify the regulation relationship between VIM and SYK in HEK293 cell by dual luciferase reporter assay system and co-immunoprecipitation (CO-IP) assay; ⑶to construct VIM wild type/variant neutrophils by lentivirus transfection method on separated mice bone marrow neutrophils, and after adding LPS, to detect the proliferation, apoptosis, phagocytosis and chemotaxis of neutrophils by CCK-8, flow cytometry and Transwell methods, respectively; to explore VIM/SYK/signal pathway by the RNA-seq, Western blot and ELISA, respectively. All above results are combined to clarify the molecular mechanism of VIM variants in NS. The project will provide a new theoretical basis for the early identification and potential drug targets of NS.