通过抑制caspase-8活性提高PRRSV体外培养滴度的初步研究OA北大核心CSTPCD

The purpose of the present study was to investigate the feasibility of promoting PRRSV replication by blocking apoptosis.Marc-145 cells were infected with a highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)strain and three apoptosis inhibitors(Z-VAD-FMK,Z-IETD-FMK and Z-LEHD-FMK)were used to respectively treat PRRSV-infected cells at different concentrations and times.At the same time,caspase-8 activity kit,quantificational real-time polymerase chain reaction(qRT-PCR)and flow cytometry were used to detect the expression of caspase-8 activity,apoptosis and pathway related factors under the best inhibitor.The results showed that z-IETD-FMK(caspases-8 specific inhibitor)was the optimal inhibitor at 10 μmol/L.Addition of 10 μmol/L Z-IETD-FMK at 48 h of PRRSV infection maximally inhibited the PRRSV titer.In addition,the caspase-8 activity of Marc-145 cells was significantly increased after PRRSV infection and,and extension of infection time caused both the apoptosis rate and proapoptotic factor Bax increased while the antiapoptotic factor Bcl-2 level decreased.After treatment with inhibitor z-IETD-FMK,the activity of caspases-8 went down and the apoptosis rate also decreased.On the other hand,the expression level of Bax was significantly lower than that in the viral infection group without inhibitor(P＜0.01).In contrast,the expression level of Bcl-2 increased,which was significantly higher than that in the viral infection group without inhibitor(P＜0.01).In conclusion,HP-PRRSV infection induced apoptosis of Marc-145 cells through caspase-8 pathway,preventing viral replication.Caspase-8 specific inhibitor effectively inhibited cell apoptosis thereby increasing the PRRSV titer.