迟缓爱德华氏菌对大菱鲆巨噬细胞相关生物效应分子产生的影响

摘要

Edw ardsiella tarda is an important facultative intracellular pathogen of fish with capability of survival and replication within macrophages.E.tarda-macrophage interactions play a very important role in the defense mechanism of fish against invading E.tarda.The mechanisms,however,are not well char-acterized.To gain insight concerning this process,characterization of macrophages response to infection with high virulent and low virulent E.tarda was explored.The macrophages were isolated by 31% —45%Percoll density gradient centrifugation from head kidney of turbot Scophthalmus maximus and maintained in L-15 medium at 20 ℃ for 24 h.The isolated cells with typical macrophage morphology were evaluated by light microscopy with Giemsa stain.The macrophages were cultured overnight before co-culture with the mid-log phase bacteria at a multiplicity of infection(MOI)of 50:1.At indicated time,caspase-3 ac-tivity,ROS and NO production of macrophages infected with high virulent and low virulent E.tarda were assayed by colorimetry,DCFH-DA and Griess method.It was found that high virulent strain significantly inhibitted the caspase-3 activity in macrophages(P<0.05).Furthermore,low virulent strain caused much higher level of ROS and NO production than high virulent strain did(P<0.05).The findings suggested that high virulent strain could follow some strategies to achieve a successful intracellular lifestyle,inclu-ding anti-apoptosis,preventing ROS and NO generation of macrophages.The data described here are ex-pected to provide valuable information in appreciating the strategies used by E.tarda to survive and to know n how intracellular survival leads to disease manifestation.Proper understanding of this mechanism can help to develop suitable strategies to overcome disease caused by E.tarda in aquaculture.%作为兼性胞内寄生菌,迟缓爱德华氏菌能够在鱼类巨噬细胞内存活繁殖.巨噬细胞与迟缓爱德华氏菌的相互作用是迟缓爱德华氏菌致病的重要环节.探讨不同毒力迟缓爱德华氏菌对巨噬细胞相关生物效应分子产生的影响,将为明晰该菌的致病机理和胞内寄生机制奠定基础.用31% /45%Percoll密度梯度离心分离大菱鲆头肾巨噬细胞,在L-15细胞培养基上20 ℃培养24 h,Giemsa染色显示培养的细胞符合巨噬细胞典型的形态特点;体外培养的巨噬细胞以感染复数50:1进行迟缓爱德华氏菌的侵染试验.采用比色法,荧光探针DCFH-DA和Griess法动态分析不同毒力迟缓爱德华氏菌感染对巨噬细胞Caspase-3活性、活性氧和一氧化氮分泌的影响.试验结果表明,迟缓爱德华氏菌感染的巨噬细胞能够检测到较高的Caspase-3活性,明显的活性氧和一氧化氮的分泌(P<0.05),但强毒株能够显著抑制巨噬细胞Caspase-3的活性(P<0.05);同时,与弱毒株相比,强毒株也表现出对巨噬细胞分泌活性氧和一氧化氮明显的抑制作用(P<0.05).研究结果提示,迟缓爱德华氏菌可能通过抑制巨噬细胞凋亡,减少活化巨噬细胞产生活性氧和一氧化氮等杀菌效应分子来达到其胞内寄生和繁殖的目的.