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Objective: 1. To observe the cytopathogenic effect of epithelial cell infected by different multiplicity and different infection time of respiratory syncytial virus, so that to set up the Optimum conditions of epithelial cell infected by respiratory syncytial virus.2. To set up the method of epithelial cell and fibroblast coculture, and simultaneously explore the level ofαsmooth muscle actin(α-SMA) and extracellular matrix(ECM) in HPBFs ,so that to better understand the mechanism of acute and subsequent recurrent wheezing associated with RSV bronchiolitis. 3. To observe the efficancy of leukotriene receptor antagonists and inhaled corticosteroids on the the level ofα-SMA and extracellular matrix in HPBFs, wish to provide the theory on choice of effective prevention and treatment measures against RSV infection.Methods:1.Culture of 16-HBE、HEp-2 cells and HPBFs, plaque technic determine infectious unit of RSV, establish the model of RSV infected 16-HBE cell in vitro. 2.MTT analytical method detect growth index of 16-HBE infected by different MOI and on different incubated days, to detect RSV infected 16-HBE by method of RT-PCR and direct immunofluorescence assay. 3. To establish the coculture model of epithelial cell and fibroblast, the level of colⅠand FN in cell supernatant was measured by an enzyme-linked immunosorbent assay(ELISA) andα-SMA measured by western blotting(WB) and indirect immunofluorescence assay(IFA). 4. To establish the coculture model of epithelial cell and fibroblast, the fibroblast incubated with graded concentration of budesonide and Montelukast, then determine the level of colⅠand FN in cell supernatant, simultaneously detect the level ofα-SMA.Results: 1. The GI of 16-HBE inoculated with 0.1 MOI after 72h was 0.85, that is not many dead cells compared with the uninfected control group, 85% of cells still living. The direct immunofluorescence technology can detect the virus antigen expression in the cytoplasm of 16-HBE, the virus nucleic acid of RSV was amplified by the PCR technology. the MOI of 0.1 and infection time of 72h is the best conditions for 16-HBE infected RSV. 2. At 24h and 48h,there is noα-SMA expression in 16-HBE-RSV+HPBFs group and 16-HBE+HPBFs group, starting from the 72h,the groups have different levels ofα-SMA expression, 96h is highest expression levels. at 72h, 96h and 120h,theα-SMA expression of 16-HBE-RSV+HPBFs group were significantly higher than the 16-HBE + HPBFs group (t=21.883,61.836,4.826, P all<0.05). In 24h, 48h, 72h, 96h and 120h , the ColⅠlevels of 16-HBE-RSV+HPBFs group were significantly higher than the 16-HBE+ HPBFs group(t=21.692,380.474,32.216,28.812,43.301, P all <0.05). the ColⅠlevels of 120h were significantly higher than 24h,48h,72h and 96h(t =61.776,40.876,54.11,33.845, P all <0.05). The Fn levels were no significant difference between 16-HBE-RSV+HPBFs group and 16-HBE+ HPBFs group. 3. Theα-SMA expression of 16-HBE-RSV+HPBFs group were significantly higher than the 16-HBE+HPBFs group (t=18.913,P<0.05), Theα-SMA expression of the 16-HBE-RSV+HPBFs+BUD(10-8) group、16-HBE-RSV+ HPBFs+ BUD(10-6) group and16-HBE-RSV+HPBFs+BUD(10-5) group were not different from 16-HBE-RSV+HPBFs group (P all>0.05). The ColⅠlevels of 16-HBE-RSV+ HPBFs group were significantly higher than the 16-HBE+HPBFs group(t= 35.694,P<0.05). The ColⅠlevels of the 16-HBE-RSV+HPBFs+BUD(10-8) group、16-HBE-RSV+HPBFs+BUD(10-6) group and 16-HBE-RSV+HPBFs+BUD(10-5) group were not different from 16-HBE-RSV+HPBFs group (P all>0.05). 4. Theα-SMA expression of 16-HBE-RSV+HPBFs group were significantly higher than the 16-HBE+HPBFs group ( t=36.957,P<0.05 ) , Theα-SMA expression of 16-HBE-RSV+HPBFs+Mont (10-7) group、16-HBE-RSV+HPBFs+Mont(10-6) group and 16-HBE-RSV+HPBFs+Mont(10-5) group were significantly lower than the 16-HBE+ HPBFs group(t=22.583、63.316 and 159.96 , P all <0.05). The ColⅠlevels of 16-HBE-RSV+ HPBFs group were significantly higher than the 16-HBE+HPBFs group(t=101.817,P<0.05). The ColⅠlevels of 16-HBE-RSV+HPBFs+Mont (10-7) group、16-HBE-RSV+HPBFs+Mont(10-6) group and 16-HBE-RSV+HPBFs+Mont(10-5) group were significantly lower than the 16-HBE+ HPBFs group(t=12.846、26.522和28.418, P all <0.05).Conclutions:1. 16-HBE were successfully infected by RSV, but infected 16-HBE cell body appears only increase, the brightness was decreased, do not form a typical integration of disease, the MOI of 0.1 and infection time of 72h is the best conditions for 16-HBE infected RSV.2. 16-HBE infected RSV promote fibroblast transformat to myofibroblasts and secrete ECM ColⅠ,so that to initiate the process of airway remodeling.3. Leukotriene receptor antagonist montelukast can inhibit the process of 16-HBE infected RSV promoting fibroblast transformat to myofibroblasts and secreting ECM ColⅠ, suggesting that CysLTs play an important role in RSV-related wheezing and airway remodeling.4. Inhaled corticosteroids Budesonide can′t inhibit the process of 16-HBE infected RSV promoting fibroblast transformat to myofibroblasts and secreting ECM ColⅠ,this may be the reason for corticosteroids not good for RSV-related infant wheezing.5. RSV infection induces airway epithelial cell secrete CysLTs, which initiates the process of airway remodeling ,this is the molecular biological mechanism of recurrent wheezing and persistent asthma in susceptible individuals. Clarifing the molecular biological mechanism of RSV-related wheezing and persistent asthma provides a theoretical basis for the selection of drugs for the effective prevention of early childhood asthma.
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