摘要
【目的】探讨猪圆环病毒2型(PCV2)感染量、感染时间与被感染RAW264.7细胞活性氧水平动态变化的相关性,为建立RAW264.7细胞氧化胁迫体外模型提供参考依据。【方法】PCV2原液调至5×106/m L,经10、102、103和104倍稀释(10-1、10-2、10-3和10-4稀释度)后,感染作用RAW264.7细胞2 h,弃病毒液,加入含5%FBS的DMEM培养液继续培养,分别于第4、8、12、24和48 h收集细胞上清液或细胞,测定一氧化氮(NO)、活性氧自由基(ROS)、还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)、黄嘌呤氧化酶(XOD)、髓过氧化物酶(MPO)和诱生型一氧化氮合酶(i NOS)等指标。【结果】10-1 PCV2感染RAW264.7细胞后能有效升高细胞NO、ROS水平,降低细胞GSH水平和GSH/GSSG,升高细胞XOD、MPO和i NOS活性,表明以10-1 PCV2感染RAW264.7细胞一定程度上能改变细胞氧化还原状态,诱导细胞产生氧化应激。【结论】PCV2感染能诱导RAW264.7细胞发生氧化应激,并确立10-1 PCV2(5×105/m L)体外感染RAW264.7细胞4~24 h是建立RAW264.7细胞氧化胁迫模型的最佳条件。
【Objective】The present study investigated the relationship between infection dose, infection time of porcine circovirus type 2（PCV2） and dynamic changes of reactive oxygen species（ROS） levels in PCV2 infected RAW264.7 cells, so as to provide a reference for the establishment of in vitro oxidative stress model of RAW264.7 cell. 【Method】 PCV2 virus liquid of 5×10^6/m L was prepared, and was 10, 10^2, 10^3 and 104times diluted（10^-1,10^-2,10^-3,10^-4 dilutions） before use.RAW264.7 cells were infected with five different concentrations of PCV2 for 2 hours, and then virus liquids were discarded. The cells were cultured in DMEM after adding 5% FBS for 4, 8, 12, 24 and 48 h. Cell supernatant or cells were collected and levels of nitric oxide（ NO）, reactive oxide species（ ROS）, reduced glutathione（GSH）, oxidized glutathione（GSSG）, xanthine oxidase（XOD）, myeloperoxidase（MPO） and inducible nitric oxide synthase（i NOS） were determined. 【Result 】After 10-1PCV2 infected RAW264.7 cells, levels of NO and ROS increased while GSH levels and GSH/GSSG ratio decreased. Activities of XOD, MPO and i NOS also increased. These findings suggested that 10-1PCV2 infection could change cellular redox state and induce cells to produce oxidative stress. 【Conclusion】PCV2 infection can induce oxidative stress in RAW264.7 cells. 10^-1PCV2（5×10^2/m L） infection in vitro on RAW264.7 cells for 4-24 h is the optimum condition for establishing oxidative stress model.
出处
《南方农业学报》
CAS
CSCD
北大核心
2017年第1期151-157,共7页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31260619)
高等学校博士学科点专项科研基金项目(20134501110004)
