Establishing inflammatory response model of RAW264.7 cells induced by pseudorabies virus in vitro

【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 levels 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.