草鱼C5a/C5aR通路确证及其炎症效应机制初探

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.

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。

脊椎动物C5a/C5aR通路在炎症反应调控中发挥了重要功能；前期研究发现草鱼C5及炎症因子ILs、IFN-γ、TNF-α和FG表达量在草鱼感染GCRV后显著增高，猜想草鱼感染GCRV病毒后是否也存在C5a/C5aR通路的炎症调控路径。首先克隆出草鱼C5 cDNA全长（NCBI登录号：MK256357.1）编码1693aa，预测草鱼补体C5为分泌信号肽，且含有过敏毒素因子的同源结构域ANATO和补体膜攻击复合体结构域C345C，两个结构域是补体因子的特征结构域，从而确定了草鱼有补体C5基因；然后，确定草鱼的C5a为C5a链的LINK序列“RLKR”之后，截止于C5转化酶的作用位点R之前共71个aa序列。构建了草鱼GCRV攻毒模型为1龄草鱼种，100g/尾，腹腔注射，最适宜发病水温为25-28℃，死亡水温为26℃。取样后对比草鱼健康和染病各组织间C5、C5a、C5aR、ILs、IFN-γ、TNF-α和FG等因子的基因表达，表明除IL-8在肠道表达最高外，其余各因子均在肝脏和脾脏中的表达量最高，并且与其他组织中的表达存在显著性差异，且在感染GCRV后12小时，组织间表达量最高，从而证明了草鱼的这些因子在同一个表达路径上；进而提出这些因子之间的调控联系假设是否也如哺乳动物一样还有磷酸化因子P38、ERK1参与了如上路径的调控，因此，制备出草鱼C5a和C5aR和F2多克隆抗体，采用WB和免疫共沉淀，以及磷酸化检测草鱼P38、ERK1蛋白表达水平的变化，结果表明，P38与ERK1蛋白与C5a、TNF-α、IL-1β和F2蛋白在GCRV中的病程中呈现同步及相似的表达规律，进而蛋白水平上确证了草鱼C5a/C5aR通路参与其被GCRV感染后的炎症调控，并且是通过P38和ERK1蛋白的磷酸化来实现的。研究结果将为草鱼出血病的综合防控和草鱼抗逆育种研究提供科学支持。