Detection Of HBV Status In Peripheral Immunocytes And Bone Marrow Hematopoietic Stem/Progenitor Cell Of HBV Related Liver Disease Patients After Liver Transplantation And Its Clinical Significance Analysis

Liver transplantation (LT) is a final and effective treatment for end stage liver diseases (ESLD) patients. In China, hepatitis B virus (HBV) related liver diseases have become the primary indication for LT. Eeven lamivudine (LAM) and hepatitis B immunoglobulin (HBIG) were combined administrated in clinic, the HBV graft reinfection and HB recurrence after LT remain still high, which severely leads a poor clinic outcomem of LT. This study aimes to research the status of a major extrahepatic HBV reservoir, namely Immume-Hematopoietic System, try to reveal the possible mechanisms of HBV reinfection and HB recurrence post-LT.In part 1, a sensitive and credible real-time fluorescence polymerase chain reaction (RT-FQ-PCR) for HBV covalently closed circular DNA (cccDNA) detection was established. In part 2, peripheral blood mononuclear cells (PBMC), T lymphocytes, B lymphocytes, monocytes and bone marrow (BM) hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC) were isolated respectively by Ficoll-Hypaque density-gradient centrifugation combine magnetic cell sorting and separation (MACS). The intracellular and sera total HBV DNA and HBV cccDNA were detected by RT-FQ-PCR. The serological markers of HBV were detected simultaneously. It proved that no evidences of HBV replication and HBV X gene integration were found, but there are HBV DNA persisting positive in immunocytes, HSC and HPC, even undergoing LAM and 1BIG combined therapy for a long time after LT. Part 1:An enzyme assistant Taq man probe real-time fluorescence quantitative PCR detection method of HBV cccDNAObjective: To establish a sensitive and specific real-time fluorescence quantitative PCR for HBV covalently closed circular DNA (HBV cccDNA) detection.Methods: According to the differences in construction between HBV relaxed circular DNA (HBV rcDNA) and HBV cccDNA, two couples of primers for HBV cccDNA detection, cccF₁-R₁ and cccF₂-R₂, were synthesized basing on attainable literatures at present. Series dilutions of HBV rcDNA which extracted from Dane particles, and plasmid DNA which extracted from pBHB4 plasmid containing a monomer of the HBV adr₄ subtype genome, were using as the templates respectively. Before and after treated by different dosage single-strand-specific mung bean nuclease (MBN), rcDNA and cccDNA were amplified by two couples of primers for HBV cccDNA respectively. The products of PCR were analyzed by 1% agarose gel electrophoresis. The optimal primers of HBV cccDNA detection and the optimum dosage of MBN were chosen for detection of HBV cccDNA, according to the results of PCR and electrophoresis.Results: 1. cccF₁-R₁ and cccF₂-R₂ can amplify not only cccDNA, but also HBV rcDNA when the titer was more than 1.92×10⁶ copies/ml and 1.92×10⁵ copies/ml respectively. In other words, the two couples of primers just possess the relative specificity for HBV cccDNA. 2. The specificity of cccF₁-R₁ excels cccF₂-R₂, because the former flanks the nick in minus strand and the gap in plus strand of HBV rcDNA but the latter only flanks the nick in minus strand. 3. MBN can promote the specificity of cccDNA primers by digesting selectively the single strand DNA present only in HBV rcDNA. The optimum dosage of MBN was 5.0IU. 4. Using ccc F₁-R₁, Taq man probe and MBN, a sensitive and specific quantification method of HBV cccDNA detection was established, with a detective range of 5.00×10²ï½ž5.00×10⁸ copies/ml.Conclusions: A sensitive and specific Taq man probe real-time FQ-PCR for HBV cccDNA quantitative detection, assisted by mung bean nuclcasc, has been established successfully, which is content with the requests of our follow research. Part 2:Quantitative detection and clinical significance of total HBV DNA and HBV cccDNA in peripheral immunocytes and bone marrow CD₃₄⁺ cells from HBV related liver disease patients after liver transplantation undergoing combined prophylaxis of LAM and HBIGObjective: The long-term clinical outcomes of liver transplantation (LT) had been reduced by HBV graft reinfection/HB recurrence. Peripheral blood mononuclear cell (PBMC), in which HBV DNA can be persistent positive, were considered the major source of HBV graft reinfection/HB recurrence post-LT. But the half-life time of PBMC is limited in some weeks to months. Undergoing the combined prophylaxis protocol of lamivudine (LAM) and hepatitis B immunoglobulin (HBIG), the HBV infected PBMC would be cleared thoroughly step by step after LT. In order to clarify why HBV DNA can persistent in PBMC for long time post liver transplantation, this study was carried out.Methods: 25 HBV related LT recipients (22 males and 3 females) were selected, whose postoperative time were ranged from 11 to 77 months (33.32±14.81 months) and age were from 33 to 61 years old (43.32±8.76 years). The combined prophylaxis protocol of LAM and HBIG has been chosen to prevent HBV graft re-infection/HB recurrence. 7 case were negative for serum HBV DNA (＜10³ copies/ml) prior to operation and 18 were positive with a mean trier of 10^{5.954±1.457} copies/ml. In the study, 20ml peripheral vein blood of every case was collected and 23 samples of 20ml bone marrow were obtained from 23 of 25 cases with written content. PBMC, T, B, monocyte (MNC) and CD₃₄⁺ cell were isolated respectively by Ficoll-Hypaque density-gadient centrifugation and magnetic cell sorting and separation (MACS). The samples of serum were collected simultaneously. The quantification of anti-HBs was performed using electrochemi-luminescence immunoassay (ECLIA). The detection of HBV markers was performed using enzyme linked immunosorbent assay (ELISA). The cellular DNA was extracted by DNA isolation and purification kit (Watsonbiot, Shanghai China) following the manufacture's instructions. The titer of HBV DNA and HBV covalently closed circular DNA (cccDNA) in serum, PBMCs, T, B lymphocytes, MNCs and CD₃₄⁺ cells, including hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), were detected using real-time fluorescence quantitative polymerase chain reaction (RT-FQ-PCR).Results: 1.25 cases are all positive for anti-HBs with a mean serum anti-HBs titer of 119.1±90.7IU/L, ranged from 12.37 to 372.6IU/L and the serum HBV antigens of all cases are negative. Anti-HBe and anti-HBc are positive in 5 cases; Anti-HBe and Anti-HBc are negative in 6 cases; Anti-HBe is positive and Anti-HBc is negative in 1 case; Anti-HBe is negative and Anti-HBc is positive in 13 cases. 2. All serum HBV DNA is negative in 25 cases. 3. Not only immunocytes including PBMC, MNCs and B, T lymphocytes, but also CD₃₄⁺ cells are all positive for HBV DNA, with geometric mean titer of 10^{3.3263±0.6956}, 10^{2.9425±0.6462}, 10^{2.7213±0.6223}, 10^{2.9522±0.932}, 10^{3.3373±0.6583} copies per 10⁶ cells respectively. And the distribution of HBV DNA in different subset cells is diversity according to the result of ANOVA. The influence factors of HBV DNA titer in different subset cells are also diversity according to the results of regression and correlation analysis. 4. There is no evidence of HBV replication in CD₃₄⁺ cells and immunocytes (no HBV cccDNA were detected in cells).Conclusions: Even combined prophylaxis of lamivudine and hepatitis B immunoglobulin after LT, the HBV related recipients are still HBV DNA positive in peripheral immunocytes and bone marrow CD₃₄⁺ cells influenced by complicated factors. Because there is no HBV cccDNA detcted in immunocytes, HBV DNA positive in CD₃₄⁺ cells may be a key factor of HBV DNA persisting in immunocytes, especially to lymphomononuclears, which may lead nonresponse to HBV vaccine and HBV graft reinfection/HB recurrence post operatively. But the mechanism of HBV transmission intercellular and the effect of intracellular HBV DNA titer on immune system have not been clarified. Further studies are needed to gain a better understanding on HBV graft reinfection. Part 3:Detection of HBV X gene integration in PBMC and bone marrow CD₃₄⁺ cells from HBV related liver disease patients using HBV-Alu-PCR after liver transplantationObjective: To clarify whether HBV X gene integrate in PBMC and bone marrow CD₃₄⁺ cells from HBV related liver disease patients after liver transplantation.Methods: PBMCs were obtained from 25 HBV related liver disease patients after liver transplantation and bone marrow CD₃₄⁺ cells obtained from 23 cases among them. The cellular DNA was extracted by DNA isolation and purification kit (Watsonbiot, Shanghai China) following the manufacture's instructions. Specific primers to HBV X gene and to human Alu repeats were used to amplify the virus integration through a 3-round hemi-nest PCR. The PCR final product was judged by 1.2% agarose electrophoresis, ligated to T vector, proliferated in E. coil 5αand sequenced.Results: According to agarose electrophoresis and sequencing analysis, there were no HBV X gene integration in PBMCs and bone marrow CD₃₄⁺ cells from HBV related liver transplant recipients after surgery.Conclusions: Because of the radical change of HBV bionomy in HBV related liver transplant recipients after operation, the fundamental condition of HBV integration has been lost, which led peripheral immunocytes and bone marrow CD₃₄⁺ cells not suit to HBV integrate to human genome.