Study On The Responses Of TLR3 And The Cytokine Of TLR3-Dependent To HBV Infection On Bewo Cell

Objective:①To explore the protein expression of Toll-Like Receptor 3 on Bewo with hepatitis B virus(HBV) infection,To study the protein expression of TLR3 on Bewo stimulated by Poly I: C (TLR3 agonists) for resisting the HBV infection.②To explore the releasing of cytokine of TLR3-Dependent on Bewo with hepatitis B virus(HBV) infection,To study the cytokine of TLR3-Dependent stimulated by Poly I:C (TLR3 agonists) on Bewo exposure to the HBV DNA.③To study the effects of TLR3 and the cytokine of TLR3-Dependent for resisting the HBV infection.Methodscultured human placenta trophoblast cells was divided into two groups, (untreated control group and stimulated group by Poly I:C);flow cytometry was conducted to measure TLR3 protein to define the influence Poly I:C stimulation for TLR3 on Bewo, then cultured human placenta trophoblast cells was divided into three groups, (untreated control group, co-cultured with HBV DNA positive serum group, co-cultured with HBV DNA positive serum group after stimulating PolyⅠ:C,Bewo and culture supernatant were selected respectively at 8h,16th, 24h,48h after co-cultured with HBV DNA positive serum, the protein of TLR3 on Bewo cell were detected by FACs, IL-2, IL-6, IL-10, TNF-α, IFN-βin supernatant were analyzed by ELISA. FQ-PCR (fluorescence quantitative PCR) was used to detect the HBV DNA on Bewo cell and in supernatant.Results(DTLR3 protein's mean fluorescence intensity of Bewo cells in PolyⅠ:C stimulated group was 53..58±3.11, statistically higher than that (42.14±3.85) in no-PolyⅠ:C stimulated group (P=0.00)②during three groups (untreated control group, co-cultured with HBV DNA positive serum group, co-cultured with HBV DNA positive serum group after stimulating Poly I:C), TLR3 protein of Bewo cell were all statistically significant((P=0.064,0.997) at the time of co-cultured time with HBV serum 8h,16h,24h or 48h, compared to untreated control group, TLR3 protein of Bewo cell co-cultured with HBVDNA positive serum were higher, no-PolyI:C stimulated group,8 h (68.67),16 h (82.98),24 h (94.08),48 h (79.66), PolyI:C stimulated group 8 h (59.85),16 h(70.45),24 h(79.18),48 h(87.27).③IL-6,IL-10,TNF-α,IFN-βof Bewo cell were all statistically higher in PolyI:C stimulated group than no-PolyI:C stimulated group at the time of co-cultured time with HBV serum 8h,16h, 24h or 48h. after exposure to HBVDNA, Production of IL-6, TNF-a by Bewo was Sharply increasing respectively exposing for 8h,24h, IL-10, IFN-βwere both increases slowly.④Both no-PolyI:C stimulated group and PolyI:C stimulated group, HBV DNA can be detected in Bewo cells co-cultured with HBV DNA positive serum for 24 hours,48hours after being washed four times by PBS. But as for two groups,when Bewo cells were cocultured with HBV serum for 8h, HBV DNA in supernatant were not not statistically significant((P=0.064,0.997). However, while the time of cocultured time with HBV serum was 16h,24h or 48h, HBV DNA in supernatant of no-PolyI:C stimulated group were all higher than that in the separate cotemporaneous group, and this difference was statistically significant with all the value of P less than 0.001. further more,the virus concentrations of HBV DNA in supernatant and PBMC decreased with the extension of time.Conclusion①hepatitis B virus can induce to increase the expression of TLR3 protein on Bewo.Thus, PolyI: C can strengthen the ability of Bewo cell co-cultured with HBV DNA positive serum to increase the expression of TLR3.②IL-6,IL-10,TNF-α,IFN-βwas secreted by Bewo cell co-cultured with HBV DNA positive serum, We established that stimulation of TLR3 positive Bewo cell lines leads to TLR3-dependent expression of IL-6 IL-10,TNF-α,IFN-βThese results Indicate that the cytokine profile of Bewol cells can be modified through TLR3 stimulation.③PolyI:C can down-regulated susceptibility of Bewo to HBV, This gives us a clues that TLR3 may influence HBV replication. Our finding suggest that TLR3 is involved in the immune responses of Bewo cells after exposure to HBV DNA and has the potential to alter the cytokine milieu and influence the outcome and consequences of HBV DNA infection...