| [Objective] To indentify the molecular forms and expression status of HBV in hepatic lymph nodes, and to investigate its role in HBV recurrence in hepatitis B related liver transplantation recipients by detecting HBV DNA, HBV cccDNA and HBsAg in lymph nodes from hepatoduodenal ligament of HBV infected patients.[Methods]Part1:To establish a detection system of HBV DNA and HBV cccDNA by PCR: Based on the characteristics of racial and geographic distribution in enrolled cases, we selected HBV genotype B, C in GenBank and compared the difference of gene sequence, then designed three pairs of nested primers in three independent areas S, C and X-region. When a sample was positive in two or three regions, we consider the sample is positive for HBV DNA. According to the structural and biological characteristics of HBV cccDNA, we firstly digested the DNA samples extracted from hepatic lymph nodes using PSAD enzyme, and we also designed the primers for cccDNA flank the gap region and the incomplete strand of DNA. pCP10plasmid was used as a positive control, we used the samples extracted from liver tissue for testing to determine the best primer annealing temperature and the detection of sensitivity.Part2:36patients(male32, females4, average age54.4±6.8) underwent liver transplantation for hepatitis B-related end-stage liver disease in our hospital from June2010to March2011who were positive for serum hepatitis B surface antigen and negative for HBV DNA quantitative detection (<500IU/mL) were involved to detect HBV DNA and HBV cccDNA in the hepatic portal lymph nodes by PCR method established in part1.Part3:From June2010to July2011,9lymph nodes were extracted from HBsAg-positive donors. To detect HBV DNA and HBV cccDNA in the hepatic portal lymph nodes in this group by the PCR method established in part1. Hepatitis B surface antigen in the lymph nodes were detected by enzyme-linked immunosorbent assay(ELISA). Immunohistochemical method was used to detect the expression of hepatitis B surface antigen in the lymph nodes.Data were analyzed by t-test and Fisher’s exact test. [Results]Part1:The detection system of HBV DNA and HBV cccDNA by PCR was specificity, less miscellaneous, and this methods can be used for resesrch.Part2:30cases from the36HBV-related liver transplantation recipients were positive for HBV DNA and the positive rate was83.3%. In three independent detection area of HBV DNA, the positive rate in S, C and X regions were86.1%(31/36),86.1%(31/36),66.7%(24/36) respectively.17cases were positive for HBV cccDNA, the positive rate was47.2%.For this population, thirty-six cases were divided into drug treatment group(22) and non-drug treatment group(14) according to the utility of nucleoside analogues. Drug treatment group was divided into two subgroups:drug treatment>3months group(14) and drug treatment≤3months group(8). The positive rate of HBV DNA: The difference was not statistically significant between drug treatment group (72.7%,16/22) and non-drug treatment group(100%,14/14)(P=0.062), the difference was not statistically significant between drug treatment>3months group(71.4%,10/14) and drug treatment≤3months group(75%,6/8) in the subgroups of drug treatment group(P=1.000). The positive rate of HBV cccDNA:Drug treatment group(22.7%,5/22) was significantly lower than the non-drug treatment group(85.7%,12/14)(P=0.000), drug treatment>3months group(7.1%,1/14) was significantly lower than drug treatment≤3months group(50%,4/8) in the subgroups of drug treatment group (P=0.039).Part3:7cases were positive for HBV DNA in9HBsAg-positive liver transplantation donors, the positive rate was71.8%. In three independent regions of HBV DNA, The positive rate in the S, C and X regions were77.8%(7/9),77.8%(7/9),66.7%(6/9) respectively. Five cases were positive for HBV cccDNA, the positive rate was55.6%. Hepatitis B surface antigen enzyme-linked immunosorbent showed that:3cases were positive, the positive rate was33.3%.(3/9), lower than HBV DNA and HBV cccDNA detection rate of hepatic lymph nodes in this group. A scattered distribution of a small number of positive cells can be observed by immunohistochemical.[Conclusions]1. The detection system of HBV DNA and HBV cccDNA by PCR was specificity, less miscellaneous, and this methods can be used for resesrch.2. HBV can infect hepatic lymph nodes of patients with CHB, hepatic lymph nodes may be one of the extrahepatie HBV reservoirs, and may be one of the factors in HBV recurrence of hepatitis B related liver transplantation patients.3. Treating with nucleoside analogues more than3months can significantly decrease the replication of HBV in hepatic lymph nodes of hepatitis B Virus-associated liver transplantation recipients.4. Hepatitis B virus expression was inhibited in hepatic lymph nodes of HBsAg-positive carries. |
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