Novel Immune-Type Receptors and Cytotoxicity In Zebrafish

在许多脊椎动物中，先天免疫系统的一项功能是由细胞毒性细胞执行的，这些细胞识别并摧毁病毒感染的细胞或癌细胞。参与这些过程的分子机制在哺乳动物自然杀伤（NK）细胞中得到了很好的研究，但对其他物种的分子机制知之甚少。最近在硬骨鱼中发现了一个具有与哺乳动物NK受体相似特征的新免疫型受体（NITR）基因家族。因此，人们假设这些基因在鱼类的细胞毒性细胞功能中起作用。该项目将确定斑马鱼NITR基因家族是否在已知具有细胞毒性的细胞系中表达，以及斑马鱼NITR表达细胞是否表现出细胞毒性活性。首先，将通过RT-PCR和RNA印迹分析确定多个NITR基因家族的组织特异性表达模式。其次，将通过RNA原位杂交在已知具有细胞毒性的细胞系中检测NITR基因的表达。第三，生成并分析斑马鱼转基因系，其中绿色荧光蛋白将作为表达nitr细胞的体内荧光生物标志物，然后将其分离并测试细胞毒性活性。本项目获得的知识将为细胞毒性信号通路的功能提供新的见解，并阐明鱼类先天免疫反应性的分子机制。该项目的结果将有助于更好地了解鱼类的免疫功能，包括那些具有农业重要性的免疫功能，并将对理解占据不同环境生态位和遇到不同病原体的生物体的细胞毒性防御的分子机制产生影响。该项目将涉及多名研究生和本科生，他们将获得各种遗传、分子和生化技术方面的专业知识，同时接受实验设计和科学过程方面的培训。

PROJECT SUMMARYIn many vertebrates, one function of the innate immune system is carried out by cytotoxic cells that recognize and destroy virally infected or cancerous cells. The molecular mechanisms involved in these processes are well studied in mammalian natural killer (NK) cells, but very little is known about them in other species. A family of Novel Immune Type Receptor (NITR) genes has recently been identified in bony fish with characteristics similar to the mammalian NK receptors. Thus, it has been hypothesized that these genes play a role in cytotoxic cell function in fish. This project will determine whether zebrafish NITR gene families are expressed in cell lineages known to possess cytotoxic properties and whether zebrafish NITR-expressing cells exhibit cytotoxic activity. First, tissue-specific expression patterns will be determined for multiple NITR gene families by RT-PCR and RNA blot analysis. Second, expression of NITR genes will be examined by RNA in situ hybridization in cell lineages known to be cytotoxic. Third, transgenic lines of zebrafish will be generated and analyzed in which the Green Fluorescent Protein will serve as an in vivo fluorescent biomarker for NITR-expressing cells, which will then be isolated and tested for cytotoxic activity. Knowledge gained from this project will provide new insight into the function of cytotoxic signaling pathways and elucidate the molecular mechanisms of innate immune reactivity in fish.The results of this project will contribute to a better understanding of immune function in fish, including those of agricultural importance and will have implications for understanding the molecular mechanisms of cytotoxic defense in organisms that occupy vastly different environmental niches and encounter diverse pathogens. This project will involve multiple graduate and undergraduate students who will gain expertise in various genetic, molecular and biochemical techniques, while being trained in experimental design and the scientific process.