任春芝 谢映红 王秋华 韦英益 胡庭俊

摘要：【目的】探讨猪伪狂犬病毒（Pseudorabies virus，PRV）感染对小鼠单核巨噬细胞（RAW264.7）炎症反应的影响，确定PRV的最佳感染剂量和感染时间，为建立RAW264.7细胞体外病毒感染炎症反应模型打下基础。【方法】PRV按10倍递增稀释成10-5～10-1 PRV稀释液，感染RAW264.7细胞并孵育1.5 h，弃病毒液后加入含5%胎牛血清的DMEM维持培养液继续培养，分别于继续培养2、4、8、12、24和48 h时收集细胞上清液，采用ELISA测定IL-6、IL-10、IL-1β、TNF-α、MCP-1和IFN-γ分泌水平及环氧合酶（COX-1和COX-2）活性，并以CCK-8法测定细胞活性。【结果】以PRV感染RAW264.7细胞4～48 h后均能通过PCR扩增获得PRV核酸的特异性条带，故选择4～48 h作为后续研究的PRV感染时间范围;10-2 PRV～10-1 PRV感染可显著降低RAW264.7细胞活性（P<0.05，下同），10-3 PRV组仅在培养48 h时出现下降趋势，而10-5 PRV～10-4 PRV感染对RAW264.7细胞活性无显著影响。PRV感染RAW264.7细胞后，其胞内炎症因子IL-6、IFN-γ、TNF-α、IL-1β和MCP-1的分泌水平整体上呈升高趋势，其中10-3 PRV感染RAW264.7细胞12 h能显著或极显著（P<0.01）提高IL-6、IFN-γ、TNF-α、IL-1β和MCP-1的分泌水平;10-4 PRV～10-1 PRV感染组的IL-10分泌水平均呈升高趋势，而10-5 PRV感染组在感染8和24 h时IL-10分泌水平明显低于空白对照组，至感染48 h所有病毒感染组的IL-10分泌水平均降低;10-3 PRV～10-1 PRV感染8～24 h能有效提高RAW264.7细胞的COX-2活性，但对COX-1活性的影响不明显。【结论】PRV感染能诱导RAW264.7细胞发生炎症反应，其中10-3 PRV体外感染RAW264.7细胞8～12 h是建立RAW264.7细胞炎症反应模型的最佳條件。该模型可应用于PRV感染与RAW264.7细胞炎症反应相关干预药物的研究，为进一步揭示PRV感染机理及开发抗病毒感染药物提供理论依据。

关键词： 猪伪狂犬病毒（PRV）;RAW264.7细胞;体外诱导;炎症反应;炎症因子;环氧合酶

中图分类号： S852.659.1                                文献标志码： A 文章编号：2095-1191（2021）05-1370-08

Abstract：【Objective】The effect of inflammatory reaction on RAW264.7 cells infected with pseudorabies virus（PRV）was investigated to establish the inflammatory response model in vitro by screening the best infective dose and infective time， and provide reference for establishing RAW264.7 in vitro virus infection inflammation model. 【Method】RAW264.7 cells were infected with five different concentrations of PRV which were diluted by serial 10 times to the concentration from 1×10-5 to 1×10-1 for 1.5 h. Then the virus liquids were discarded. Subsequently， the cells were plated in DMEM with 5% calf serum and then cell supernatants were collected after cultured for 2， 4， 8， 12， 24 and 48 h respectively. The le-vels of interleukin 6（IL-6）， interleukin 10（IL-10）， interleukin 1β（IL-1β）， tumor necrosis factor Alpha（TNF-α）， monocyte chemoattractant protein-1（MCP-1），interferon-γ（IFN-γ） and the activities of both cyclooxygenase 1（COX-1） and cyclooxygenase 2（COX-2） were determined by ELISA kits. The activities of cells were detected by CCK-8 method. 【Result】The specific bands of PRV nucleic acid were obtained by PCR amplification at 4-48 h post PRV infection in RAW264.7 cells， so 4-48 h was selected as the time range of PRV infection in the subsequent studies. The activities of RAW264.7 cells were significantly decreased in 10-2 PRV-10-1 PRV groups and only trended to decrease at 48 h in 10-3 PRV group（P<0.05， the same below）， however， there was no significant influence on the RAW264.7 cells activities in 10-5 -10-4 PRV groups. The secretion of inflammatory factors such as IL-6， IFN-γ， TNF-α， IL-1β and MCP-1 were increased in general after PRV infected RAW264.7 cells. 10-3 PRV infected RAW264.7 cells 12 h could significantly or extremely significantly（P<0.01） increase secretion levels of IL-6， IFN-γ， TNF-α， IL-1β and MCP-1. The secretion level of IL-10 was increased in 10-1 PRV-10-4 PRV groups， while notably lower than that in the blank control group at 8 and 24 h after infection in 10-5 PRV group， and the level was decreased at 48 h in all of the PRV infected groups. The secretion level of COX-2 was  increased for 8-24 h post infection in 10-3 PRV-10-1 PRV group， but there had no obvious effect on the secretion level of COX-1. 【Conclusion】PRV infection induces inflammatory reaction in RAW264.7 cells. 10-3 PRV infection in RAW264.7 cells in vitro for 8-12 h is considered as the optimal condition for the establishment of the inflammatory response model. This model can be applied to the study of PRV infection and inflammatory response in RAW264.7 cell which relates to the intervention action of drugs， and the results provide theoretical basis for reveal the mechanism of PRV infection and the development of anti-viral infection drugs.

Key words： pseudorabies virus （PRV）; RAW264.7 cell; in vitro induction; inflammatory response; inflammatory factor; cyclooxygenase

Foundation item： National Natural Science Foundation of China（32072907，31560708）

0 引言

【研究意义】猪伪狂犬病毒（Pseudorabies virus，PRV）可引起多种家畜和野生动物发病，猪是该病毒的唯一自然宿主，仔猪感染死亡率接近100%，给全球养猪业带来重大经济损失（Pontes et al.，2016;梁祺英等，2017）。PRV感染新生仔猪可导致其神经系统紊乱甚至死亡，感染怀孕母猪可造成流产，感染成年猪则引发呼吸系统疾病，且急性感染存活的猪群将终生带毒（Zhang et al.，2019）。感染PRV的猪群通过分泌物和排泄物传播病毒，主要通过直接接触传播，也可通过水和污染物传播。PRV疫苗免疫是预防猪伪狂犬病最有效的方法，Bartha-K61疫苗可有效控制PRV传播（华涛等，2019），但无法防治PRV变异毒株引起的感染。自2011年以来，我国部分接种Bartha-K61疫苗的养猪场仍然暴发猪伪狂犬病（An et al.，2013;Freuling et al.，2017），因此急需加强PRV感染致病機理研究，以寻找理想的抗病毒感染药物。……