Abstract:

Objective To construct a respiratory syncytial virus（RSV） infected the model of air-liquid interface（ALI）cultured human bronchial epithelial cells（hBEC）,and to provide a cell model closer to the in vivo environment for further study of respiratory virus pathogenesis. By analyzing the effect of RSV infection on the expressions of high mobility group box protein 1（HMGB1） and phosphorylated mixed-lineage kinase domain-like protein（pMLKL）,to investigate the injury pathogenesis caused by RSV. Methods The hBEC was inoculated on Transwell membrane and cultured unsubmerged mode. After the confluence of 100%,cells were transferred to ALI culture. After the cells were differentiated and matured,they were infected respectively with RSV according to the following groups:6 h control group,6 h MOI 1.0 group,6 h MOI 3.0 group,24 h control group,24 h MOI 1.0 group,24 h MOI 3.0 group,each group with 3 repeated holes. The effect of RSV infection on the expressions of HMGB1 and pMLKL was confirmed by immunofluorescence staining. Results After 4-10 d of submerged culture,the cell confluence reached 100%. Then,cells were transferred to ALI interface culture. After about 4 weeks,the distribution of cell bands became clearer,and visible mucus was secreted to form a mucus layer. HE staining showed typical pseudostratified epithelium,and immunofluorescence staining showed that RSV successfully infected cells at MOI 3.0 24 h. Anti-HMGB1 and anti-pMLKL immunofluorescence staining showed that under the condition of infection,there was pink fluorescence in the nucleus [blue 4',6-diamidino-2-phenylindole（DAPI）and red HMGB1 fluorescence merged results],which confirmed the expression of HMGB1 after RSV infection. However,there was no pMLKL expression neither before nor after RSV infection. Conclusions The well-differentiated hBEC can be obtained by submerged and ALI culture,which maintain cell morphology and function for a long time,and provide a cell model for respiratory virus infection and other common respiratory disease studies. At MOI 3.0 24 h,RSV successfully infects the cell model and causes the expression of HMGB1.

Key words: Human bronchial epithelial cell, Air-liquid interface culture, Respiratory syncytial virus, High mobility group box protein 1, Phosphorylated mixed-lineage kinase domain-like protein