Establishment of an Inflammatory Model of RAW264.7 Cells Infected with H6N6 Subtype Avian Influenza Virus
The correlation between infection amount and time of H6N6 subtype avian influenza and the dynamic changes of inflammatory level in infected RAW264.7 cells were investigated in the study to provide a reference for establishing an inflammatory model of RAW264.7 cells in vitro.The virus titer(TCID50)was calculated by Reed-Muench method.RAW264.7 cells were infected with 100 TCID50 of H6N6 subtype avian influenza virus for 2 hours.The virus venom was discarded and the culture was continued by adding DMEM medium containing 5％FBS.The culture supernatant or cells were collected at 12,24 and 48 h,respectively,and the cell viability was determined by CCK8.The ultrastructure of cells was observed by transmission electron microscope.The levels of interleukin-1 β(IL-1β),interleukin-2(IL-2),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in mice were detected by ELISA.The mRNA expression levels of IL-1β,IL-2,IL-4,IL-6 and TNF-α were detected by qRT-PCR.The results showed that the cell viability decreased significantly after H6N6 infection(P＜0.01),mitochondria were swollen,autophagosomes were increased,and the contents of IL-1β,IL-2,IL-6 and TNF-α were signifiicantly increased(P＜0.01),the mRNA expression levels of IL-1β,IL-2,IL-4,IL-6 and TNF-α were significantly increased(P＜0.01),and reached the peak at 24 h after infection.The results showed that the RAW264.7 cell model infected with H6N6 subtype avian influenza virus in vitro was successfully constructed,which would provide conditions for further study of its pathogenic mechanism.
万方数据
维普
