斑马鱼npsn基因在中性粒细胞中的作用机理

Neutrophils play important roles in innate immune system mainly dependent on various enzyme-containing granules to kill the engulfed microorganisms. Zebrafish nephrosin (npsn) was specifically expressed in the neutrophils but the function is largely unknown. Here, we generated a transgenic reporter line Tg(npsn:eGFP)smu7, in which green fluorescent protein (GFP) expression was driven by the npsn promoter. Detailed characterization of Tg(npsn:eGFP)smu7 fish confirmed that the neutrophil lineage was specifically marked by GFP expression suggested that npsn was predominantly expressed in neutrophils rather than in macrophages. We generated an npsn mutant (npsnsmu5 and npsnsmu6) by utilizing CRISPER/Cas9 system to investigate the in vivo function of Npsn. The overall development and the number of neutrophil in npsnsmu5 was not changed in Npsn deficient mutants, while the neutrophil anti-bacterial function is defect in this mutant. When infected with Escherichia coli (E. coli), the npsnsmu5 mutants showed a lower survival rate than WT siblings did. These findings demonstrate that zebrafish Npsn promotes the host defense against bacterial infection. In this study, we generated an npsn-deficient zebrafish mutant and established the Tg(npsn:EGFP)smu7 transgenic line, which provided a useful tool for studying neutrophil development and behavior during host wound healing or defense against infection in vivo. In this proposal, we are going to elucidate the genetic and molecular relationship between npsn and regulatory factors during neutrophil development, and reveal the mechanism of npsn in innate immunity of zebrafish.

中性粒细胞是血液中为数最多的一种白细胞，能够防御病原体入侵，抵抗感染，是非特异性宿主防御系统中的重要防线。中性粒细胞消除病原体的主要方式为通过分泌因子招募吞噬细胞或自身对病原体吞噬消除。而中性粒细胞的吞噬功能主要依赖于细胞内的颗粒以及颗粒酶。Nephrosin是存在于斑马鱼中性粒细胞中的一种水解酶，是颗粒酶的组成部分，但其功能至今仍不明确。我们的前期工作分别利用Tol2转座系统以及CRISPER/ Cas9系统获得了Tg(npsn:eGFP)smu6可特异标记中性粒细胞转基因斑马鱼与移码突变的斑马鱼突变体npsnsmu5和npsnsmu6。且npsn smu5斑马鱼表现为大肠杆菌的抵御能力下降。本项目将进一步研究Npsn在中性粒细胞中的作用机制，与已知因子的分子互作及遗传学调控关系，并通过细菌感染实验明确Npsn对中性粒细胞抗菌作用的影响以及相关分子机制，揭示Npsn在非特异性免疫中的作用

生物体的髓系细胞分化是一个动态而有序的过程。骨髓中的造血干细胞首先分化成为髓系祖细胞，然后再分化成为粒细胞和单核/巨噬细胞。其中粒细胞分为嗜酸性粒细胞、嗜中性粒细胞和嗜碱性粒细胞，由于髓系细胞的广泛分布和重要作用，当髓系细胞功能缺陷时会引起多种疾病。如当中性粒细胞缺陷时可引起急慢性白血病、免疫缺陷症等，因此构建这些疾病的动物模型，对了解该疾病的致病机理以及临床治疗具有非常重要的指导意义。本文以斑马鱼为模型建立了中性粒细胞缺陷感染与炎症模型。我们选取了一种特异性表达在斑马鱼中性粒细胞中的颗粒酶Nephrosin（Npsn），为了研究Npsn在斑马鱼中性粒细胞中的作用，我们首先利用CRISPER/Cas9技术构建了npsn的突变体（npsnsmu5）,在npsn缺陷体中，中性粒细胞的发育和数量没有显著性的改变，但是中性粒细胞的抗菌能力显著下降。在感染大肠杆菌（Escherichia coli）后，相对于野生型斑马鱼，npsnsmu5突变体的生存率显著下降，体内清除细菌能力显著减弱。并且过表达npsn后，npsn过表达斑马鱼的存活率显著升高。这些结果显示，Npsn能够提高斑马鱼抵抗大肠杆菌感染的能力。并且npsn缺陷和过表达斑马鱼可以作为研究体内固有免疫的斑马鱼模型。