| BackgroundIt is well known that hepatitis B virus (HBV) infection is very common in Southeast Asia and Africa, and the HBV related end-stage liver diseases become the main indication for liver transplantation in these regions especially in China. For these patients transplanted for HBV related end-stage liver diseases, one of the most important obstacles in achieving long-term satisfied outcome is HBV recurrence. When HBV recurrents, the graft hepatitis will rapidly progress to severe cirrhosis and the liver function will deteriorate sharply and eventually lead to patient death or liver retransplantation. The risk factors of HBV recurrence are numerous, including immunosuppressants usage, preoperative HBV replicative status, HBV genome type and the HBV recurrence prophylaxis therapy, etc. The immunosuppression therapy strategy in liver transplantation can be divided into two sorts: the CsA-based immunosuppressant regimens and the FK506-based immunosuppressant regimens.The multicenter studies conducted in USA and Europe showed that FK506 was superior to CsA in concerns of acute and chronic rejection, patient and graft survival, etc. For patients with hepatitis C, one multicenter study showed that the patient survival was better in FK506 group than which in CsA group;another study demonstrate that the hepatitis C recurrence probability and serum viral RNA load were both lower in FK506 group than that in CsA group. Up to date, few studies were performed with respect to the effect of CsA or FK506-based immunosuppression therapy regimen on HBV recurrence.HepG2.2.15 cell line, a HepG2 human hepatoma cell line derivative which permanently transfected with a plasmid containing two head-to-tail dimers of the HBV genome, can not only release high level of HBsAg and HBeAg into supematants, but also support the assembly and secretion of replicative intermediates of HBV DNA and Dane particles during culture, due to this, the HepG2.2.15 cells can be regarded as an approving in vitro model for HBV associated research work. In this study, we evaluate the effects of CsA and FK506 on HBV replication in order to find out the proper immunosuppressive protocol in the HBV positive recipients after liver transplantation, and further achieve long-term survival of these patients after liver transplantation.MethodsCell viability activity test of MTT assay was used to identify the nontoxic concentrations of CsA and FK506 to the cultured HepG2.2.15 cells. The concentration of CsA or FK506 would be regarded as nontoxic if the corresponding cell viability activity was no less than 95 percent, using this method, the nontoxic concentrations of CsA and FK506 were confirmed. The HepG2.2.15 cells were treated with different nontoxic concentrations of CsA (Q-20.0 ug/ml) or FK506 (0 ~1 OOng/ml). The culture mediums were collected every 24 hours, and the hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in supernatant were detected by ELISA analysis, and the suppression rates of HBsAg and HBeAg were calculated. After treated by CsA or FK506 for 4 days, the cells were gathered and the intracellular DNA was extracted, then the HBV DNA replication levels were analyzed by slot blot hybridization. The HBV replication level was evaluated by ELISA analysis of HBsAg and HBeAg, and by slot blot hybridization analysis of HBV DNA.All the detection items in this study were repeated at least 3 times, and the results were expressed as mean+SD. The One-way ANOVA was used to compare the difference between control and every treated group;the Spearman correlation test was used to analyze the correlation between reagents' concentrations and HBV replication levels. All statistic analysis was performed with SPSS 11.5 software and /'-value <0.05 was considered as significant.ResultsThe MTT assay showed that the cell viability activity was no less than 95 percent when treated by CsA at concentrations of less than 40.0ng/ml, or by FK506 at concentrations of less than 400ng/ml. So we confirmed that the nontoxic concentrations of CsA and FK506 were 0 - 40.0ug/ml and 0 ~ 400ng/ml, respectively. CsA at different nontoxic concentrations could suppress the expression of HBsAg and HBeAg, and inhibit the HBV DNA replication in a dose-dependent manner, the Spearman correlation test showed that positive relationship existed between CsA concentrations and antigen suppression rates and negative relationship existed between CsA concentrations and HBV DNA replication levels. After the treatment of CsA at the concentrations of 1.3, 2.5 and 5.0|ig/ml for 4 days, thesuppression rates of HBsAg were(16.5 ± 9.4)%, (21.5 ±8.9)% and(33.1 ±5.3)%, respectively;the suppression rates of HBeAg were(7.8 ± 2.2)%, (11.0 ±2.3)% and(20.8± 1.5)%, respectively;compared to the control, the relative HBV DNA replication level were (56.1 ± 16.5)%, (41.7 ± 10.9)% and (39.5 ± 9.5)%, respectively. FK506 at different nontoxic concentrations has no effect on the suppression rates of HBsAg and HBeAg, and the HBV DNA replication level.ConclusionCsA can dose-dependently inhibit the HBV replication in vitro, while FK506 does not have similar activity. |
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