| Objective:A relevance study between HBVcccDNA and Th1/Th2 in different phases of chronic HBV infection, we try to explore the relationship between different immune status and HBVcccDNA.Methods:Hepatic biopsy specimens were obtained from 40 patients with HBV infection and 10 normal controls. The cases were divided into 11 cases of immune tolerant phase, 11 cases of immune clearance phase, 8 cases of inactive phase and 10 cases of reactive phase. According to Proposal of Prevention and Treatment for Viral Hepatitis amended in the Tenth National Academic Conference for Viral Hepatitis in 2000.Real-time PCR were used to detect HBVDNA, HBV- cccDNA in sera and liver biopsies. Immunohistochemistry (IHC) was employed to study the disposition of IFN-γand IL-10 in liver biopsies.Results:1. Correlation between quantification of hepatic tissue HBVcccDNA and the expression of IFN-γ, IL-10 in hepatic tissue in different phases of chronic HBV infectionThere was a negative correlation between HBVcccDNA and IFN-γin immune clearance phase, reactive phase, r=-0.693, P<0.05; r=-0.666, P<0.05.There was no correlation in immune tolerant phase either inactive phase, P>0.05.There was a positive correlation between HBVcccDNA and IL-10 in immune tolerant phase, immune clearance phase, inactive phase and reactive phase, r=0.866, P<0.01; r=0.693, P<0.05; r=0.744, P<0.05; r=0.701, P<0.05.2. Correlation between quantification of hepatic tissue HBVcccDNA and the expression of IFN-γ, IL-10 in hepatic tissue when ALT≥2×ULN and 40≤ALT<2×ULNThere was a negative correlation between HBVcccDNA and IFN-γwhen ALT≥2×ULN, r=-0.828,P<0.05. There was no correlation between HBVcccDNA and IFN-γwhen 40≤ALT<2×ULN, P>0.05.There was a positive correlation between HBVcccDNA and IL-10 when ALT≥2×ULN, r=0.828, P<0.05. There was no correlation between HBVcccDNA and IL-10 when 40≤ALT<2×ULN, P>0.05.3. The expressions of IL-10, IFN-γin hepatic tissueUnder light microscope, there was no expression of IL-10 in control and mild expression in chronic HBV infection, and the cells with IL-10 had brown and yellow kytoplasm and cell membrane. The expression of IL-10 was significantly enhanced in different phases of chronic HBV infection than that in control, (32.27 vs 8.00, 27.64 vs 8.00, 32.00 vs 8.00, 28.00 vs 8.00) P<0.01. There were no significant differences between different phases of chronic HBV infection.There was no expression of IFN-γin control and mild expression in chronic HBV infection, and the cells with IFN-γhad brown and yellow kytoplasm and cellular nucleus. The expression of IFN-γwas significantly enhanced in different phases of chronic HBV infection than that in control, (21.14 vs 10.00, 39.18 vs 10.00, 21.25 vs 10.00, 34.15 vs 10.00) P<0.01. The expression of IFN-γwas enhanced in immune clearance phase and reactive phase, and significantly enhanced in the area of cellular necrosis. The expression of IFN-γwas significantly enhanced in immune clearance phase and reactive phase than immune tolerant phase and inactive phase P<0.01.There were no significant differences between immune clearance phase and reactive phase, immune tolerant phase and inactive phase.In immune tolerant phase the expression of IL-10 was significantly enhanced than IFN-γ, P<0.05; in immune clearance phase the expression of IFN-γwas significantly enhanced than IL-10, P<0.05; There were no significant differences in reactive phase and inactive phase.4. Quantification of hepatic tissue HBVDNA and HBVcccDNA in different phases of chronic HBV infection Quantification of hepatic tissue HBVDNA was 6.68×10~2 copies/mg-5.40×10~7copies/mg and hepatic tissue cccDNA was 1.0copy/mg-8.88×10~5copies/mg. The hepatic tissue HBVDNA in immune tolerant phase, immune clearance phase, inactive phase and reactive phase were all positive while the positive cases of the hepatic tissue HBVcccDNA in each group were 11/11, 11/11, 5/8, 9/10.The average rank of quantification of hepatic tissue HBVcccDNA in immune tolerant phase, immune clearance phase, inactive phase and reactive phase were 32.09, 26.00, 10.63, 9.60, and HBVcccDNA in each group were 30.45, 28.00, 6.94, and 12.15. Hepatic tissue HBVDNA, compared with the inactive phase and reactive phase, immune tolerant phase and immune clearance phase increased obviously P<0.01; but there were no statistic differences between immune tolerant phase and immune clearance phase, P>0.05; and also no statistic differences between inactive phase and reactive phase P>0.05.The results of HBVcccDNA were the same with HBVDNA.5. Quantification of sera HBVDNA in different phases of chronic HBV infectionThe positive cases of sera HBVDNA in immune tolerant phase, immune clearance phase, inactive phase and reactive phase were 11/11, 11/11, 0/8, 10/10. Quantification of sera HBVDNA was 1.00 copy/ml-7.41×107copies/ml. The average ranks of quantification of sera HBVcccDNA in each group were 28.36, 30.55, 4.50, and 13.60. Compared with the inactive phase, immune tolerant phase, immune clearance phase and reactive phase increased obviously, P<0.01; Compared with the reactive phase, immune tolerant phase and immune clearance phase increased obviously, P<0.01. 6. Correlation between quantification of hepatic tissue HBVcccDNA and quantification of sera and tissue HBVDNA in chronic HBV infection.There was a strong correlation between HBVcccDNA and HBVDNA level in the tissue (r=0.856, P<0.001), There was a strong correlation between the HBVcccDNA and HBVDNA level in the sera (r=0.842, P<0.001), There was also a positive correlation between the HBVDNA level in the tissue and HBVDNA level in the sera (r=0.779, P<0.001).Conclusion:1. The different phases of chronic HBV infection have different immune status, companying the different relationship with HBVcccDNA in liver tissue.The expression of IFN-γwas significantly enhanced in immune clearance phase and reactive phase than immune tolerant phase and inactive phase, there was a negative correlation between HBVcccDNA and IFN-γ, with the increase of IFN-γthe HBVcccDNA is lower. That IFN-γin the immune clearance phase and reactive phase participate the clearance of HBVcccDNA. There was a positive correlation between HBVcccDNA in the tissue and IL-10 in immune tolerant phase, immune clearance phase, inactive phase and reactive phase. HBVcccDNA and IL-10 increased consistency show the increase in IL-10 may be associated with the persistent infection of chronic hepatitis B virus.The expression of IFN-γwas significantly enhanced when ALT≥2×ULN than 40≤ALT<2×ULN,(16.17 vs 8.93),P<0.05. meanwhile there was a negative correlation between cccDNA and IFN-γwhen ALT≥2×ULN, we think that the immune resonse has started, antiviral therapy can be carried out.2. The chronic HBV infection has Th1/Th2 imbalance. Mainly in immune tolerance phase and immune clearance phase.Th2 dominate in immune tolerance phase; Th1 dominate in immune clearance phase.3. Compared with the quantification of hepatic tissue HBVcccDNA, hepatic tissue HBVDNA and sera HBVDNA we found that in inactive phase all of sera HBVDNA were negative, all of HBVDNA were positive in tissue, 5/8 HBVcccDNA were positive, show when the level of sera HBVDNA is low enough not to be detected, we cannot casually infer hepatic tissue HBVDNA and HBVcccDNA level from sera HBVDNA level.The level of quantification of hepatic tissue HBVDNA and HBVcccDNA may reflect HBV infection and duplication in the body directly. And so it is more reliable than levels of sera HBVDNA. |
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