草鱼C5a/C5aR通路确证及其炎症效应机制初探

高等脊椎动物补体系统中的C5a/C5aR通路普遍存在，且在炎症反应调控过程中发挥了重要功能；草鱼出血病是由GCRV感染并伴随显著炎症反应的重大危害疾病。在GCRV感染条件下的草鱼脾脏转录组研究不仅证实了草鱼存在补体系统，而且发现草鱼补体因子C5、FG在GCRV感染后，C5a被大量激活、炎症因子ILs、IFN-γ、TNF-α和FG表达量显著提高的有趣现象。基于草鱼这一类似高等动物C5a/C5aR通路调控的表现，本项目拟采用病理组织学、原位杂交和流式细胞技术，在组织学和分子水平确证草鱼在GCRV感染条件下C5a/C5aR炎症调控通路的存在；并采用q-PCR和western-blot技术检测通路中各基因和蛋白的表达，初步解析草鱼C5a/C5aR通路在草鱼出血病中的炎症调控机制；以期为草鱼抗逆性育种研究新思路奠定科学基础，为草鱼出血病的综合防控提供技术依据。

Complement system C5a/C5aR pathway is an important regulatory factor during inflammation in all mammals. Grass carp hemorrhagic disease is caused by grass carp reovirus (GCRV) and is responsible for tremendous losses of grass carp (Ctenopharyngodon idella) production. Preliminary studies suggesting C5a is activated in grass carp infected with hemorrhagic disease have been supported by gene expression analysis. Previously, we obtained the whole cDNA sequence of complement component 5(C5) and fibrinogen(FG) as well as incorporated fragments from both genes into an eukaryotic expression vector. To further understand the response of the grass carp (Ctenopharyngodon idella) to GCRV, our objectives are to certify the function of C5a/C5aR pathway in grass carp by observing mortality over several days in infected and uninfected grass carp, and performing histopathological examination, C5a protein expression analysis in peripheral blood, and C5aR gene expression analysis in spleen and liver tissues by determining whether or not macrophage is activated; study the response of infected grass carp in vivo as well as in vitro, using grass carp spleen cells under three treatment conditions: infected, uninfected, and infected with C5a interferented，use q-PCR assays to determine gene expression of ILs, IFN-γ, TNF-α, FG, and fibrogen, use western-blot to detect protein expression of C5a and FG, and use flow cytometry determine if macrophage is activated. The proposed study will help to elucidate the C5a/C5aR pathway regulatory mechanism in grass carp during grass carp hemorrhagic disease and provide a theoretical foundation for prophylaxis and treatment, which is of scientific importance and has valuable industrial applications.