The Role Of Toll-like Receptor 3 In Apoptosis Of Bewo Cells Induced By Hepatitis B Virus

ObjectiveTo explore whether hepatitis B virus can replicate in bewo cells to set up a susceptible cell model for infection experiments, whether HBV can induce apoptosis of bewo cells, and study the role TLR3 may play in apoptosis induced by hepatitis B virus.Methods①PCR was used to detect HBV cccDNA expression of bewo cells which were cultured with HBV positive serum.②F low cytometry,tarnsefarse-mediated dUTP nick end-labeling were used to detect apoptosis rate of bewo cells to indentify the right dose of hepatitis B virus ,appropriate infection time with which bewo cells can be successfully stimulated apoptosis.③on the basis of this,the influence Poly I: C stimulation had on the apoptosis of bewo cells co-cultured with HBV DNA positive serum was observed,and flow cytometry was conducted to measure TLR3 protein to explore the role of TLR3 in induction of HBV to bewo apoptosis.Results①H BV cccDNA can be detected in bewo cells co-cultured with HBV DNA positive serum for 48 hours, which were cultured another 24 hours, 48hours after being washed six times by PBS.②when the bewo cells were cocultured with 50μl HBV serum for 8h, 24h, 48h, early apoptotic rates of these cells were respectively 4.34±3.42%,1.98±1.42%,6.58±3.44%,the total apoptotic rates were respectively 10.78±5.36%,7.21±2.39%,16.35±9.61%. The difference of early apoptotic rates and the total apoptotic rates between HBV group and control group was not statistically significant with all the value of P more than 0.05.③w hen bewo cells were cocultured with 100μl HBV serum for 8h, early apoptotic rates(5.55±1.80%) and total apoptotic rates (10.88±2.33%) were not statistically significant((P=0.064, 0.997). However, while the time of cocultured time with HBV serum was 24h or 48h, early apoptotic rates(20.76±2.13%, 24.23±6.87%), total apoptotic rates(25.41±1.93%, 24.92±12.71%) and apoptotic indexes(25.58±2.11%,30.95±9.02%) were all higher than that in the separate cotemporaneous group, and this difference was statistically significant with all the value of P less than 0.001.④A fter stimulated by PolyI:C,early apoptotic rates (6.28±3.70%,6.25±1.35%) and total apoptotic rates (7.69±3.71%,6.67±1.54%) of the bewo cells co-cultured with HBV serum for 24h or 48h were significantly decreased with all the value of P less than 0.05.⑤TLR3 protein's mean fluorescence intensity of bewo cells in PolyI:C stimulated group was 36.0217±2.71701, statistically higher than that (32.5852±1.40918) in no-PolyI:C stimulated group(P=0.02).Conclusion①Bewo cells can be used as a good model for HBV infection experiments in vitro, because hepatitis B virus can replicate in them.②hepatitis B virus can induce apoptosis of bewo cells, increase the expression of TLR3 protein.Thus, TLR3 may mediate the apoptosis of bewo induced by HBV.③PolyI: C can increase the expression of bewo TLR3 protein, decrease apoptosis rate of bewo cells co-cultured with HBV DNA positive serum. The activation of TLR3 by PolyI: C may inhibit apoptosis of bewo stimulated with HBV.