| ObjectiveTo study gene mutations in basic core promotor (BCP) and precore regions of HBV DNA and Th1/Th2 immunological state in patients with chronic severe hepatitis B. To explore the relationship between gene mutations in basic core promotor (BCP) and precore regions of HBV DNA and severe hepatitis and the pathogenesis of severe hepatitis.Methods1. A cross-sectional study was tested between 38 patients with chronic severe hepatitis B and 40 patients with chronic hepatitis B(CHB).The nucleotide segments of basic core promotor (BCP) and precore regions of HBV DNA were amplified by polymerase chain reaction(PCR) from serum of these patients. Then the PCR products were sequenced directly. The serum level of IFN-γ, IL-2, IL-4 and IL-10 of these patients and 20 healthy blood donors were measured by ELISA.2. A control study was tested on 6 follow-up patients from CHB to chronic severe hepatitis B. The nucleotide segments of basic core promotor (BCP) and precore regions of HBV DNA were amplified by PCR from 2 serum specimens in the period of CHB and chronic severe hepatitis B respectively. Then the PCR products were sequenced. The serum level of IFN-γ, IL-2, IL-4 and IL-10 of these patients were measured by ELISA.3. From a patient with liver transplantation, the nucleotide segments of basic core promotor (BCP) and precore regions of HBV DNA from 3 serum specimens in the period of CHB, before and in the period of chronic severe hepatitis B respectively and 1 hepatic tissue specimen were amplified by PCR. Then the PCR products were cloned and sequenced. The serum level of IFN-?, IL-2, IL-4 and IL-10 of these patients were measured by ELISA and the hepatic tissues were check up by pathobiology and immunohistochemistry (IHC).Results1. Spot-mutations were found in 69 of 78 amplified gene segments of basic core promotor (BCP) and precore regions of HBV DNA. The five more mutation sites were nt1764(48.7%), 1762(38.5%), 1896 (26.9%), 1766(17.9%) and 1862(14.1%). The mutations of A1762T /G1764A, G1896A, G1862T and A1762T/G1764A /G1896A were observed in 42.1%, 36.8%, 21.1% and 23.7% chronic severe hepatitis B cases and in 15.0%, 17.5%, 7.5% and 7.5% CHB cases. The differences in incidence rates between two groups were significant (p<0.05). The mutations of A1762T/G1764A, G1896A,G1862T and A1762T/G1764A/G1896A were observed in 64.7%,58.8%,35.3% and 41.2% chronic severe hepatitis B c bewteen ases with HBeAg(-) and in 23.8%, 19.0%, 9.5% and 9.5% chronic severe hepatitis B cases with HBeAg(+). The differences in incidence rates of A1762T/G1764A, G1896A and A1762T/G1764A/ G1896A between two groups were significant (p<0.05). The mutations of A1762T/G1764A, G1896A,G1862T were found in five of six follow-up patients from CHB to chronic severe hepatitis B and A1762T/G1764A was often observed.2. The serum level of IFN-? and IL-2 in chronic severe hepatitis B group was significantly higher than that in CHB and control groups, and serum level of IL-4 and IL-10 were lower than that in CHB group (P <0.05). Compared with none mutation in basic core promotor (BCP) and precore regions of HBV DNA group, the serum level of IFN-? and IL-2 increased in the mutation group (P<0.05). The differences of IL-4 and IL-10 level between in the mutation group and nonemutation group were not significant (P>0.05). The differences of IFN-γ, IL-2 and IL-4 and IL-10 level within different mutation groups were not significant (P>0.05). To 6 follow-up patients, in the period of cluonic severe hepatitis B, the IFN-γ level were higer but IL-4 and IL-10 level were lower than that in the period of CHB respectively (P <0.05).3. A case revealed that in serum, the number of gene mutations and quasispecies of basic core promotor and precore regions in the period of CHB was relatively less than that in the period of chronic severe hepatitis B. Gene mutations in basic core promotor and precore regions of HBV DNA increased significantly in the period of chronic severe hepatitis B, especially the mutations of A1762T/G1764A and G1896A. Histochemical stain in the relic hepatic tissue manifested that HBcAg predominantly localized in kytoplasm with the progress of hepatitis, IFN-γ increased gradually, but IL-4 and IL-10 decreased to some extent.Conclusion1. The gene mutations in basic core promotor and precore regions of HBV DNA might possibly be associated with chronic severe hepatitis B. Gene mutations in basic core promotor and precore regions of HBV DNA in chronic HBV infection might aggravate the state of illness or lead to chronic severe hepatitis B. The causal relation between gene mutations and chronic severe hepatitis B was still to be verified.2. There was Th1/Th2 disturbance in cluonic severe hepatitis B and the increase of Th1 type cytokine was predominant. The gene mutations in basic core promotor and precore regions of HBV DNA may lead to increase of Th1 type cytokine and aggravate immunologic injury in liver because of affecting the synthesis and secretion of HBeAg.
|
PDF Full Text Request