柔嫩艾美耳球虫在鸡巨噬细胞中的培养及子孢子感染巨噬细胞细胞因子的mRNA水平  

Cultivation of Eimeria tenella in Chicken Macrophages and mRNA Levels of Macrophage Cytokines Infected by Sporozoites

作　　者：蒋保余 王文青 曲自刚[2] 肖星星[2] 杨顺利[2] 刘保红[2] 曾巧英[1] 蔡建平[2] JIANG Bao-yu;WANG Wen-qing;QU Zi-gang;XIAO Xing-xing;YANG Shun-li;LIU Bao-hong;ZENG Qiao-ying;CAI Jian-ping(Collage of Veterinary Medicine,Gansu Agricultural University,Lanzhou,Gansu,730070,China;State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary parasitology of Gansu Province/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural,Lanzhou,Gansu,730046,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《动物医学进展》2020年第11期33-40,共8页Progress In Veterinary Medicine

基　　金：中国农业科学院科技创新工程专项(2014-LVRI-09)。

摘　　要：以鸡巨噬细胞(HD11)为柔嫩艾美耳球虫(Eimeria tenella)的宿主细胞,将E.tenella子孢子体外接种HD11细胞,观察其入侵细胞、发育及诱导细胞抗感染免疫情况。用荧光探针和real-time PCR方法分别检测细胞内NO和ROS的含量和E.tenella感染相关的8种宿主细胞因子(IL-1β、IL-6、IL-10、IL-12p40、IFN-γ、TNF-α、iNOS和IRF7)mRNA水平。结果显示,E.tenella子孢子成功入侵HD11细胞,但并没有发育迹象。与不刺激组相比,活子孢子刺激后HD11细胞内NO含量在4 hpi显著升高,8 hpi达到最高并且随时间变化不再降低,而ROS含量在2 hpi显著升高,6 hpi达到最高并且随时间变化逐渐降低。与不刺激组相比,活子孢子刺激HD11细胞IL-1β和iNOS mRNA表达水平在6 hpi显著增加(P<0.05),IL-6、IL-10、IL-12p40、IFN-γ、TNF-α、iNOS和IRF7在10 hpi显著增加(P<0.05)。与不刺激组相比,热灭活子孢子刺激后HD11细胞IL-6和IFN-γmRNA表达水平在4 hpi显著增加(P<0.05),IL-1、TNF-α和iNOS在6 hpi显著增加(P<0.05),而IL-10、IL-12和IRF7在10 hpi显著增加(P<0.05)。表明巨噬细胞抗球虫的免疫反应中,细胞因子的显著上调可能与机体抑制球虫发育增殖或清除球虫感染有关。Chicken macrophage(HD11)was used as the host cell of E.tenella,and E.tenella sporozoites were inoculated into HD11 cells in vitro to observe the invasion cells,development and induction of cell anti-infective immunity.Fluores-scence probe and real-time PCR method were used to detect the contents of NO and ROS in cells and 8 host cytokines related to E.tenella infection(IL-1β,IL-6,IL-10,IL-12p40,IFN-γ,TNF-α,iNOS and IRF7)mRNA levels.E.tenella sporozoites successfully invaded HD11 cells,but showed no signs of development.Compared with the non-stimulated group,the NO content in HD11 cells increased significantly at 4 hpi after live sporozoite stimulation,reached the highest at 8 hpi and no longer decreased with time,while ROS content increased significantly at 2 hpi and reached the highest at 6 hpi,and gradually decreased with time.Compared with the non-stimulated group,live sporozoites stimulated the expressions of IL-1βand iNOS mRNA in HD11 cells at 6 hpi significantly(P<0.05),IL-6,IL-10,IL-12p40,IFN-γ,TNF-α,iNOS and IRF7 increased significantly at 10 hpi(P<0.05).Compared with the non-stimulated group,the expression levels of IL-6 and IFN-γmRNA in HD11 cells were significantly increased at 4 hpi after heat-inactivated sporozoite stimulation(P<0.05),and IL-1,TNF-αand iNOS were significantly increased at 6 hpi.(P<0.05),while IL-10,IL-12 and IRF7 increased significantly at 10 hpi(P<0.05).It is shown that in the immune response of macrophage against coccidia,heat inactivated sporozoites induce cell reaction faster than live sporozoites,and significant upregulation of cytokines may be related to the body's clearance of coccidial infection or inhibition of developmental proliferation.

关 键 词：柔嫩艾美耳球虫 鸡巨噬细胞 细胞因子 一氧化氮 活性氧 抗感染免疫 

分 类 号：S852.723[农业科学—基础兽医学]