The Role Of TLR3 Gene In HBV Infection And Cleaning Of BeWo Cells

Objective①To explore the relation between the protein expression level of Toll-Like Receptor 3 on BeWo cells and hepatitis B virus(HBV) infection and reproduction after HBV activated Bewo cells that express different TLR3 level.②To explore the relation of the releasing of related cytokine of TLR3-Dependent on BeWo of different state of TLR3 gene by infecting hepatitis B virus(HBV) and HBV infection and reproduction on BeWo.MethodsPEGFP-Genesil-tlr3 plasmids inhibited TLR3 gene expression were transfected into BeWo by using liposome transfection reagent Lipofectamine 2000, and 40ug/ml PolyI:C stimulated BeWo cells to improve TLR3 protein expression, then HBV infected every group. This study has four groups: control BeWo group, HBV infected plasmid (pEGFP-Genesil-tlr3) transfection group, HBV infected normal BeWo cells group, Poly I:C and HBV stimulated BeWo cells group. These cell groups were co-cultured with HBV DNA positive serum at 8h,16th,24h,48h.The four groups BeWo cells and culture supernatant were collected respectively.The expression of TLR3 protein on BeWo cells was assessed by fluorescence activated cells sorting(FACs), the HBV DNA copies on BeWo cells culture supernatant were detected by Fluorescence quantitative polymerase chain reaction, and the secreted content of cytokine IL-10,TNF-α,IFN-βin supernatant were analyzed by enzyme-linked immunosorbent assay(ELISA).Results①TLR3 protein's mean fluorescence intensity of BeWo cells in PolyI:C stimulated group was 65.11±2.73,statistically higher than that (53.27±4.45) in normal control group(t=4.536,P<0.05).②Approximately 50.24% BeWo cells were transfected when Lipofecamine 2000 was used to deliver TLR3-specific siRNA.③When normal BeWo cells group, PolyI:C stimulated BeWo cells group, and plasmid (pEGFP-Genesil-tlr3 )transfected BeWo cells group were respectively infected by hepatitis B virus (HBV), TLR3 protein of every group BeWo cells were all statistically significant((P<0.05) compared to control group at the time of co-cultured time with HBV serum 8h,16h,24h or 48h.TLR3 protein of normal BeWo cells and PolyI:C stimulated BeWo cells co-cultured with HBV DNA positive serum were higher. TLR3 protein's mean fluorescence intensity of normal control group was 8h(52.88), 16h(53.78), 24h(55.07), 48h(54.70); HBV stimulated normal cells group 8h(62.50), 16h(69.65), 24h(71.78), 48h(73.49); PolyI:C and HBV stimulated group 8h(77.27), 16h(85.36), 24h(91.26), 48h(88.27), and TLR3 protein content reached to peak value at 24h;But TLR3 protein of plasmid transfected BeWo cells co-cultured with HBV DNA positive serum was lower, 8h(46.19), 16h(45.96), 24h(46.34), 48h(49.55).④HBV DNA copies of every group cells cultured supernatant between three groups(HBV stimulated normal BeWo cells group, PolyI:C and HBV stimulated BeWo cells group and HBV stimulated and plasmid transfected BeWo cells group) were all statistically significant((P<0.05) at the time of co-cultured time with HBV serum 8h,16h,24h or 48h, and HBV DNA copies were lower with the time extension of HBV stimulation. Compared with HBV stimulated normal BeWo cells group, HBV DNA copies of PolyI:C and HBV stimulated BeWo cells group were lower,and HBV DNA copies of HBV stimulated and plasmid transfected BeWo cells group were higher.⑤IL-10,TNF-α,IFN-βof BeWo cells were all statistically higher(P<0.05) in PolyI:C stimulated group than HBV stimulated normal group at the time of co-cultured time with HBV serum 8h,16h,24h or 48h;these were all statistically lower(P<0.05)in HBV stimulated and plasmid transfected group than HBV stimulated normal group. With the time extension of HBV's stimulation, the expression level of TNF-αstarted to rise at 8 hours later and to decline at 24 hours later. The expression levels of IL-10 and IFN-βrose slowly, but the expression of PolyI:C and HBV stimulated group rose higher at 16 hours later.Conclusion①Hepatitis B virus can inducethe expression of TLR3 protein on BeWo cells, PolyI:C can strengthen the ability of BeWo cells co-cultured with HBV DNA positive serum to increase the expression of TLR3 protein, and TLR3 suppression BeWo cells had lower expression of TLR3 protein with HBV DNA positive serum, and HBV DNA copies of every group cell cultured supernatant became lower with the increasing of TLR3 protein expression. This demonstrated that TLR3 can reduce infection and reproduction of HBV.②The ability of BeWo cells were enhanced to secrete cytokine IL-10, TNF-α, IFN-βby stimulating HBV, and these cytokines were secreted weakly by TLR3 suppression BeWo with HBV. These results demonstrated that TLR3 prevented HBV infection by stimulating BeWo cells to secrete cytokines related tosignal transduction pathways.