Novel Immune-Type Receptors and Cytotoxicity In Zebrafish

在许多脊椎动物中，先天免疫系统的一种功能是由细胞毒性细胞执行的，细胞毒性细胞识别并破坏病毒感染或癌细胞。在哺乳动物自然杀伤（NK）细胞中，这些过程中涉及的分子机制得到了很好的研究，但在其他物种中却知之甚少。最近在硬骨鱼中发现了一个新的免疫型受体（NITR）基因家族，其特征与哺乳动物NK受体相似。因此，有人假设这些基因在鱼类细胞毒性细胞功能中发挥作用。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特性的细胞系中表达，以及斑马鱼NITR表达细胞是否表现出细胞毒性活性。首先，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。第二，将通过已知具有细胞毒性的细胞系中的RNA原位杂交来检查NITR基因的表达。第三，将产生并分析斑马鱼的转基因系，其中绿色荧光蛋白将用作表达NITR的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。本项目的研究成果将为鱼类细胞毒信号通路的功能提供新的认识，并阐明鱼类先天免疫反应的分子机制。本项目的成果将有助于更好地了解鱼类的免疫功能，包括那些在农业上有重要意义的，并将对理解占据截然不同的环境生态位的生物体中细胞毒性防御的分子机制产生影响。并遇到各种病原体。 该项目将涉及多名研究生和本科生，他们将获得各种遗传，分子和生物化学技术的专业知识，同时接受实验设计和科学过程的培训。

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.