Effect of lentivirus expressing TLR4 shRNA on function of rat alveolar macrophage

Objective To construct the replication-incompetent lentivirus expressing rat TLR4 shRNA,and to observe its inhibitory effect on TLR4 expression in alveolar macrophage and on lipopolysaccharide (LPS)-induced release of IL-1β,IL-6.Methods We designed and constructed four shRNA expressing plasmids with silencing effect,then they were co-transfected into HEK293T cells with the TLR4 eukaryotic vector constructed previously;the best shRNA was selected to be packed into lentivirus;and the titer was determined.The packed lentvirus was used to infect the alveolar macrophage(NR8383) in presence of LPS,and the LPS-induced IL1β,IL-6 expression was examined by ELISA.Results The best shRNA was successfully screened out and was correctly inserted into the lentivirus.The titer of the recombinant lentivirus was 2.0 × 106 TU/ml.The LPS-induced expression of IL-1β,IL-6 in the alveolar macrophages was greatly reduced after virus infection (P 0.05).Conclusion We have successfully constructed the recombinant lentivirus expressing rat TLR4 shRNA,which has an satisfactory inhibitory effect against expression of IL-1β,IL-6,paving a way for studying the chronic rejection reaction after rat lung transplantion in vivo.