The Relationship Between CccDNA Of HBcAb-positive Donor Liver Tissue And Hepatitis B Recurrence After Liver Transplantation

[Objective] To indentify the relationship between HBV cccDNA and postoper-ative recurrence of HBV in HBcAb positive liver donors by detecting HBV cccDNA in liver from HBcAb positive liver donors.[Methods]Part1:To establish a detection system of HBV cccDNA by fluorescence quantitative PCR. Based on the characteristics of racial and geographic distribution in enrolled cases,we selected HBV genotype B,C in GenBank about the complete genome sequence of50and compared the difference of HBV rcDNA and HBV cccDNA, then designed HBV cccDNA primers and probe in relatively conservative sequence. According to the structural and biological characteristics of HBV cccDNA, we firstly digested the DNA samples extracted from liver using PSAD enzyme, and we also designed the primers for cccDNA flank the gap region and the incomplete strand of DNA and probe where extend to binding sites, outer exonuclease activity will be used to cut the probe and generate a fluorescent signal.pCP10plasmid was used as a standard curve, beta-actin was used as an internal. we used the samples extracted from liver tissue for testing to determine the best primer annealing temperature and the detection of sensitivity.Part2:The relationship between HBV recurrence and HBV cccDNA of HBcAb positive liver tissue by fluorescence quantitative PCR.85of1200patients(male65, females20, average age44.55±12.37, average follow-up32.12±10.06months) underwent liver transplantation for hepatitis B-related end-stage liver disease in our hospital from January2007to January2010who were involved to detect HBV cccDNA in the donors by PCR method established in part Ⅰ.Divided into the experimental group (HBcAb positive,HBsAg negative liver, n=40), control group (HBsAg positive liver, n=15), normal group (donor normal n=30). Inclusion criteria:1)18-65years old, male or female;2) HBsAg or HBsAg and HBV DNA positive;3) signed the informed consent;4) Patients are expected to survive for more than6months after surgery. Exclusion criteria:1) serological is HBsAg-negative in preoperative;2) Patients are expected survival for less than6months;3) other hepatitis viruses are mixed infection;4) HBV virus mutates;5) severe drug allergy. All patients are applied for the treatment combine nucleoside drugs with HBIG in the intraoperative and postoperative. Test HBV serology and HBV DNA in order to confirm HBV recurrence. the rate of hepatitis B recurrence were calculated. To indentify the relationship between HBV cccDNA and postoperative recurrence of HBV in HBcAb positive liver donors.Statistical comparisons were performed using the Statistical Program for Social Sciences (SPSS16for Windows, SPSS). Categorical variables were tested using the chisquared test. Statistical significance was assumed for P-values<0.05.[Results]Part1:The detection system of HBV cccDNA in donor liver by luorescence quantitative PCR was specificity, and this methods can be used for resesrch.Part2:85cases of patients were enrolled. Respectively, the experimental group、the control group and the normal group were12,11and nothing cases of cccDNA detection positive, the positive rate was30%(12/40),73.3%(11/15), O.There was a statistically significant difference through the positive rate of HBV in these three groups (x2=8.419P=0.004).The rate of HBV recurrence in three group were10%(4/40),80%(12/15) and3.3%(1/30). The difference of HBV recurrence between the experimental group and control group was statistically significant(x2=25.913P＜0.001). There was no statistically significant between the experimental group and normal group(x2=1.149P=0.284).Respectively, the rate of HBV recurrence in the experimental group of cccDNA (+)and cccDNA (-) were7.5%(3/40),2.5%(1/40), there was a statistically significant to the HBV recurrence rate(x2=4.286P=0.038).[Conclusions]1. The detection system of HBV cccDNA in donor by luorescence quantitative PCR was builded and this methods can be used for resesrch in paraffin embedded donor liver tissues. 2. The cccDNA in HBcAb positive donor may be one of the risk factors in HBV recurrence of hepatitis B related liver transplantation patients.