| PART I HMGB1EXPRESSION EV HEPG2.2.15CELLS AND PROMOTE HBV REPLICATIONObjective:HMGB1is one of the cytokines,which plays an important role in the pathogenesis of various viral diseases recent years found,but it’s role in HBV infection has not be reported.This research detected the expression of HMGB1in HepG2.2.15cells,and the HBV DNA,HBsAg,and HBeAg detected after stimulate HepG2.2.15cells with rhHMBGl,in order to explore the function of HMGB1in CHB and provide theoretical basis for the pathogenesis of CHB.Methods:The expression of HMGB1in HepG2and HepG2.2.15cells at gene and protein levels were detected by real-time PCR and Western blot;We used free-HMGB1medium(control group)and medium contained HMGB1(100μg/L,HMGB1stimulation group) cultured HepG2.2.15cells for0h,12h,24h,36h,48h,72h and96h respectively.The expression of HBsAg and HBeAg in supernatant were detected by Abbott chemiluminescence, and the levels of HBV DNA in supernatant were detected by Real-Time PCR. Results:The expression of HMGB1mRNA and protein in HepG2and HepG2.2.15cells were detected,and the expression of HMGB1in HepG2.2.15were significantly higher than that of HepG2.The expression of HBV DNA,HBsAg and HBeAg increased significantly from24to96h after HMGB1stimulated(P<0.05),and the expression were significantly higher than that of the control group at the same piont of time(P<0.01).Conclusions:The expression of HMGB1in HepG2.2.15cells was higher than in HepG2cells,which indicated that HMGB1related to infection of HBV; Stimulated the HepG2.2.15cells with HMGB1promoted the secretion of HBV DNA,HBsAg and HBeAg,which indicated that HMGB1promoted the replication of HBV;Continuous stimulation with HMGB1could activate the replication of HBV further,HMGB1could play an important role in replication of HBV. PART11THE MOLECULAR MECHANISMS OF HMGB1PROMOTE HBV REPLICATIONObjective:The research of the first part showed that stimulated HepG2.2.15cells with HMGB1can promote HBV replication in vitro,but the molecular mechanism is still unclear. We detected the expression of toll-like receptor signaling pathway related molecular and the expression of HNF4α after stimulated HepG2.2.15cells with HMGBl,in order to explore the possible mechanisms of HMGB1in promotion HBV replication.This may find a new target for treatment of CHB.Methods:HepG2.2.15cells stimulated with HMGB1(100ug/L) and cultured for0h,12h,24h,36h,48h,72h and96h,the expression of TLR2and TLR4were detected by FCM;The expression of Myd88,phospho-JNK, phospho-ERK,phospho-p38,and NF-κB p65protein were detected by Western blot;The expression of HNF4a was measured by Real-time PCR and Western blot;The content of NF-κB and HNF4a were observed by immunofluorescence/confocal microscopy.Results:FCM showed that both TLR2and TLR4expressed in HepG2.2.15cells,and the expression of TLR4is higher than that of TLR2.The expression of TLR2had no significantly changed but the expression of TLR4was abundant from12h to96h after HMGB1stimulated;Western blot suggested that Myd88,phospho-JNK, phospho-ERK,phospho-p38,and NF-κB p65protein expressed weakly before stimulation,but increased gradually after HMGB1stimulated;The expression of HNF4a at gene and protein levels increased gradually after HMGB1stimulated,and increased significantly from48h to96h; Immunofluorescence/confocal microscopy showed that NF-κB translocated gradually from the cytoplasm to the nucleus from12h to48h after HMGB1stimulated;HNF4a fluorescence mainly located in nucleus in HepG2.2.15cells,the green fluorescence of HNF4a enhanced gradually after HMGB1stimulated.Conclusions:The Signaling pathway of HMGB1stimulate HepG2.2.15cells was mainly mediated by TLR4;The expression of HNF4a in HepG2.2.15cells was up-regulated by HMGB1stimulation,and the expression of HNF4a have a positive correlation with the expression of HBV DNA;HMGB1could activate the HNF4a through the TLR4-MyD88-MAPK-NF-κB signaling pathway and promote HBV replication. |
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