| Objective: To find the basis of RSV infection and immune response in patients with COPD,and to explore the possible role of TLR3/4 in the immune response induced by RSV infection in BEAS-2B cells.Methods: Three different groups(normal controls,SCOPD and AECOPD patients)contributed thirty sputum specimens each,and RSV in every sputum specimens was detected using nested PCR.ELISA was used to detect the expressions of TNF-α,IL-13,IL-32 and INF-β protein in sputum of each group.RT-PCR was used to detect the expressions of TLR3/4 mRNA in sputum.BEAS-2B and Hep-2 were cultured in vitro,RSV was amplified in Hep-2 and virus titer was calculated by RT-PCR.After BEAS-2B cells being infected with RSV in different groups(Control group,UV-RSV group,Different concentrations)in different time periods(12h,24 h,48h),the morphological changes of the cells were observed under a light microscope.RT-PCR was used to verify RSV and RT-PCR was used to detect the expressions of TLR3 and 4 mRNA in BEAS-2B cells stimulated by RSV.The expressions of IFN-β,IL-13,IL-32 and TNF-α in the cell supernatant were detected by ELISA.Results: RSV was not detected in the 30 sputum specimens from normal controls.In 30 sputum specimens from patients with SCOPD,3 cases of RSV were detected,accounting for 10%.In 30 sputum specimens from patients with AECOPD,8 cases of RSV were detected,accounting for 26.6%.The protein expressions of INF-β,IL-13,IL-32 and TNF-αin the sputum specimens from patients with COPD were higher than those from normal controls(P<0.05).The protein expressions of INF-β,IL-13,IL-32 and TNF-α in sputum of RSV positive group were higher than that of RSV negative group(P<0.05).The expressions of TLR3/4 mRNA in sputum of COPD patients were higher than that of normal controls(P<0.05).The expressions of TLR3/4 mRNA in sputum of RSV-positive group were higher than that of RSV-negative group(P<0.05).After RSV infected Hep-2cells,the cells were deformed from irregular polyhedral to fragmented and eventually detached,which was called cytopathic effects(CPE),Amplification of RSV in Hep-2 cellswas verified using RT-PCR.During RSV infection with different concentrations in different time periods,it was detected by ELISA that the protein expressions of INF-β,IL-13,IL-32 and TNF-α in BEAS-2B cells infected with RSV increased in comparison with the control group(P<0.05).The expressions of TLR3/4 mRNA in the RSV-infected group were significantly higher than that in the control group(P<0.05),detected by RT-PCR.Conclusion: The immune response induced by RSV in patients with chronic obstructive pulmonary disease and BEAS-2B cells may be involved in the occurrence and development of COPD. |
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