Expression Of Apoptosis Associated Factors In Peripheral Blood Mononuclear Cells Of Patients With HBV Infected And The Relationship With Chronicity Of Hepatitis B

Background and ObjectiveHepatitis B virus can easily lead to chronic infection, but the pathogenesis of chronity remains unclear. The present studies show that the apoptosis of peripheral blood lymphocytes and immune tolerance are related to the chronicity of HBV infection. Activation-induced cell death means that the actived mature lymphocytes (T cells or B cells) appear apoptosis after stimulated by active signals (especially TCR/CD3 complex), which is a physiological mechanism used to maintain the body’ s immune stability, and may be the important reason of immune tolerance. Peripheral blood mononuclear cells are mainly composed of lymphocytes, and mononuclear macrophages and natural killer cells are also included, which play an important role in the body’s immunes response, cell immune response especially.Cell apoptosis is normal physiological cell death, which is a step-by-step lysis process with the participation and strict control of all kinds of cytokines, and is used to maintain the body’s homeostasis. The exact mechanism of cell apoptosis remains unclear so far. According to the difference of initianal actived caspase, the activation of apoptosis pathways can be divided to 3 kinds:the first one is death receptor pathway, named also intrinsic pathway, which is touch off by the binding of FAS ligands and FAS(CD95). Then it can active caspase 8, subsequently active effectors such as caspase 3,6,7. The second is mitochondrial pathway,named also extrinsic pathway:when stimulated by apoptosis signals, CytC、AIF are released from mitochondrial intermembrane space and then induced apoptosis. CytC is released from intermembrane space to cytoplasm, then with the existence of ATP/dATP,CytC and Apaf-1formulate poly-complex,which can raise and active caspase-9 by N-terminal caspase recruitment domain(CARD) of Apaf-1,then lead to apoptosis. The third is endoplasmic reticulum pathway, endoplasmic reticulum stress induces the expression of caspase-12, and lead to caspase-7 transferring from plasm to endoplasmic reticulum. Caspase-7 activate caspase-12, then the latter can shear caspase-3,wich can lead to cell apoptosis.This study is aimed to study the different apoptosis pathway of peripheral blood lymphocytes and the roles it plays in the chronity of HBV infection.Methods1. Inclusion criteria and exclusion criteria:the study include 9 chronic asymptomatic HBV carriers,48 chronic hepatitis B patients (14 mild patients,22 moderate patients,12 severe patients) and 24 liver cirrhosis patients. The diagnosis of all patients are in accordance with Chronic Hepatitis B Prevention Guide in 2010 and excluded liver injuries caused by hepatitis A、C、D、E and other reasons.20 healthy people act as normal control.2. Specimen collecting:collecting fasting venous blood 7mL, anticoagulating with EDTA.3mL specimen centrifuge (2000r/min,5min) and get plasma, which stored in-80℃.4mL specimen is used to extract PBMC through Ficoll density gradient centrifugation, then stored in -80℃. Extract total RNA, measure RNA concentration and purity:thaw the frozen PBMC in ambient temperature,5min, add 1mL Trizol in it, then extract total RNA according to reagent instruction strictly and measure RNA concentration and purity, guarantee A260/280 range from 1.8 to 2.0.3. Detect the expression of FAS, Caspase-8, CYCS, Caspase-9, Caspase-3 in PBMC through real time fluorescent quantatitive PCR.4. Detect the expression of Caspase-8, Caspase-9, Caspase-3 in plasma by double antibody one step enzyme linked immunosorbent assay.Results1. The results of real time fluorescent quantatitive PCR show that the expression of apoptosis-related genes (FAS, Caspase-8, CYCS, Caspase-9, Caspase-3) in CHB and ASC groups is higher, as compared to NC and LC groups (P<0.05), LC group is higher than NC group (P<0.05), CHB and ASC groups have no statistical difference (P>0.05).2. The results of ELISA show that the concentration of Caspase-9, Caspase-8, Caspase-3 in plasma from high to low order is CHB and ASC groups、LC group、NC group, the difference is significant statistically (P<0.0083), CHB and ASC groups have no statistical difference (P>0.0083).3. The concentration of Caspase-9, Caspase-8 and Caspase-3 in plasma in HBV-DNA positive group is higher than negative group (P<0.05).4. The concentration of Caspase-9, Caspase-8 and Caspase-3 in plasma from high to low order is CHB (mild)、CHB (moderate)、CHB (severe) groups, the difference is significant statistically (P<0.01).ConclusionsHBV infection can induce the apoptosis of peripheral blood mononuclear cells, extrinsic pathway and intrinsic pathway are all involved. The apoptosis of PBMC play a role in the chronicity of HBV infection.