Effects Of Fas-siRNA On Fas/FasL Dependent Apoptosis And Inflammation Of Type â…¡ Alveolar Epithelial Cells Induced By LPS In Rats In Vitro

Objective In this study,through the establishment of LPS-induced cultured rat alveolar typeâ…¡epithelial cells injury model.To investigate feasibility of Fas-siRNA transfection in alveolar typeâ…¡epithelial cells.To investigate effects of Fas-siRNA on apoptosis and inflammatory response of typeâ…¡alveolar epithelial cells.Methods Alveolar typeâ…¡epithelial cells were isolated from adult male SD rats,purified and incubated.The cells were randomly divided into five groups:AECâ…¡group, AECâ…¡+siRNA-213 group,AECâ…¡+siRNA-641 group,AECâ…¡+siRNA-830 group, AECâ…¡+negative control siRNA group.Transfection efficiency was observed after 6h.Then Fas mRNA was measured by RT-PCR after 48h.Alveolar typeâ…¡epithelial cells were randomly divided into 5 groups:groupâ… control;groupâ…¡LPS;groupâ…¢LPS+FasL;groupâ…£FasL;groupâ…¤LPS+FasSiRNA+FasL.In groupâ…¤Fas-SiRNA were transfected 20h before LPS and FasL were added to cells.In all groups the final concentration of LPS and FasL were 10ug/ml and 5ng/ml.The cells were cultured for another 48h.Then Fas mRNA was measured by RT-PCR.Fas protein and p-caspase8 were measured by Western blot.The apoptosis rate of AEâ…¡cells were detected by flow cytometry.The concentration of MCP-1 and TNF-a were mearsured by ELISA.Result 1,efficiency of siRNA Transfection was more than 80%.In AECâ…¡+siRNA-213 group,AECâ…¡+siRNA-641 group and AECâ…¡+siRNA-830 group,FasmRNA was decreased compared with AECâ…¡group and AECâ…¡+negative control siRNA group.In AECâ…¡+siRNA-641 group,FasmRNA was in the minimum.2,Fas mRNA and Fas protein were increased in groupâ…¡,â…¢compared with control group.There were no significant difference in Fas mRNA and Fas protein between control and group(â…£,â…¤).Fas mRNA and Fas protein were decreased in groupâ…¤compared with groupâ…¡.The apoptosis rate of AEâ…¡cells, p-caspase8,the concentration of MCP-1 and TNF-a were increased in groupâ…¡,â…¢,â…£,â…¤compared with control group;The apoptosis rate of AEâ…¡cells,p-caspase8, the concentration of MCP-1 and TNF-a were increased in groupâ…¢compared with groupâ…¡.The apoptosis rate of AEâ…¡cells,p-caspase8,the concentration of MCP-1 and TNF-a were decreased in groupâ…¤compared with groupâ…¢.Conclusion AEâ…¡cells were transfected by Fas-siRNA successfully.Fas-siRNA decreased the apoptosis rate and inflammatory response of ATâ…¡cells induced by LPS were increased after Fas/FasL activation.