| Nowadays, there are about 3.5~4.0 hundred million people worldwide chronically infected with hepatitis B virus (HBV), and 1% of these patients would developed chronic severe hepatitis B (SHB) each year. Researches on the injury of liver caused by HBV revealed that T cell immunity divided into two categories. The first one is delayed hypersensitivity reaction, controlled by MHC-II molecules, its effector cell named CD4+ T-helper lymphocyte induced injury of hepatocyte through inflammatory reaction caused by releasing much types of lymphokines. The second one is T cytotoxicity reaction, controlled by MHC-I molecules, its effector cell named CD8+ cytotoxic T lymphocyte (CTL), induced injury of hepatocyte through releasing porforin, granzyme and lymphokine. Notably, most of the lymphocytes infiltrated in the necrosis area in liver of patients with hepatitis B are CD8+ CTL; it indicates that cytotoxic reaction caused by CD8+ CTL plays an important role in immunopathological injury of liver. So, there need at least three determinative factors to induce hepatocyte injury: a great quantity of CD8+ T cell infiltrated in liver, cellular membrane of hepatocyte express hepatitis B core antigen(HBcAg)and MHC-I molecule.A series of evidences indicate that destruction of virus infected hepatocyte caused by MHC class I restricted T-cell mediated cytotoxicity is the main cause of liver injury, but there still remains dispute about whether virus-specific CD8+ T cell mediated immune response is the main cause of immunologic liver injury in severe hepatitis. Peripheral blood and animal experiments found that increased degree of CD8 mRNA in liver coincided with the increased degree of ALT in serum, but there still exists controversy about the relationship between virus-specific CD8+ T cell and ALT in serum. Due to lack of sample and limitation of experimental technique, researches on the pathogenesy of severe hepatitis B confined to peripheral blood and animal experiments. There lacks profound explorations about the relationship between T cell subpopulation in liver and immunologic liver injury, so it is hard to reveal the real status of immune response in liver.Previous studies about immunologic liver injury of viral hepatitis mainly focused on the relationship between the quantity and function of immunocytes and immunopathology of liver, few researches concerned regulation of immunocytes'secretion and killing ability. Recent researches found that potassium channel and calcium channel play an important role in regulation of lymphocytic immune function. Ca2+ enters T lymphocyte through the calcium-release-activated Ca2+ channel (CRAC), which activates T lymphocyte as second messenger. K+ efflux through potassium channel forms T cell membrane potential, which influences Ca2+ influx through CRAC. Up to now, there have no studies concerning the relationship between immunologic liver injury of severe hepatitis B and potassium channel and calcium channel.In this experiment, we choose the hepatic tissues from patients with severe hepatitis B as research sample. Firstly, we analyzed the distribution of CD4, CD8 and virus-specific T cell, and we selected CD8+ T cell which most related with liver injury as target cell. Secondly, we focused on the potassium channel and calcium channel in the T cell membrane, and compared the characters of the two ion channels in CD8+ T cell membrane through gene expression, protein expression and electrophysiology. Thirdly, through medicine intervention, we explored the relationship between potassium channel, calcium channel and cell proliferation, activation, killing ability of CD8+ T cell.Main results1. Up to October 31, 2007, we have collected 77 fresh human hepatic tissues which included: 5 samples from patients with acute severe hepatitis B, 24 samples from patients with chronic severe hepatitis B, 31 samples from patients with chronic hepatitis B and 17 samples from healthy people. Some of these tissues were fixed with 4% paraformaldehyde for histological research, the remaining samples were used to extract total lymphocyte after enzymatic digestion, and the lymphocyte were stored in liquid nitrogen.2. Results of immunohistochemistry and flow cytometry indicated that: compared with healthy people, the quantity of CD8+ T greatly increased in liver of patients with severe hepatitis B, and the quantity of CD4+ T and virus-specific CTL didn't show significant difference. To identify the relation between immunocyte and liver injury, we conducted correlation analysis between experimental data and hepatic function index. We found that the quantity of CD8+ T and level of serum ALT had significant positive correlation in liver of patients with severe hepatitis B (r=0.563, p=0.021), and the quantity of virus-specific CTL and hepatic function index had no significant correlation (P >0.05).3. Calcium-activated potassium channels (KCa) showed up-regulated expression in CD8+ T lymphocyte of patients with severe hepatitis B. Results of quantitative PCR and western-blot showed that, compared with groups of normal control and chronic hepatitis B, the level of mRNA and protein of KCNN4/KCa3.1 channel increased significantly in the group of severe hepatitis B (P<0.05). Results of whole-cell patch clamp recordings showed that, compared with normal control, outward K+ current of KCNN4/KCa3.1 channel in CD8+ T cell increased significantly in the group of severe hepatitis B and chronic hepatitis B (P<0.05), and the increased level of outward K+ current in the group of severe hepatitis B significantly higher than that in the group of chronic hepatitis B (P<0.05).4. Results of laser confocal scanning microscope indicated that intracellular Ca2+ of CD8+ T cell increased largely (phenomenon of calcium overload) in the group of severe hepatitis B. Results of whole-cell patch clamp recordings showed that, compared with normal control, inward Ca2+ current of CRAC channel in CD8+ T cell increased significantly in the group of severe hepatitis B and chronic hepatitis B (P<0.05), and the increased level of inward Ca2+ current in the group of severe hepatitis B significantly higher than that in the group of chronic hepatitis B (P<0.05).5. Results of MTT cell proliferation and killing ability experiment indicated that, the degree of cell proliferation and killing ability of CD8+ T cell in group of severe hepatitis B is higher than that in group of chronic hepatitis B. Results of medicine intervention experiment found that, the ability to influence cell proliferation and killing ability as follows: Antagonist of CRAC > KCa channel blocker > Kv channel blocker, which indicated that calcium channels have larger influence than the potassium channels on the ability of CD8+ T cell.From the above results, we can draw such conclusions: activity and non-specific killing ability of CD8+ T cell in liver of patients with severe hepatitis B are higher than that in liver of normal control, and CRAC calcium channels and KCNN4/KCa3.1 potassium channels maybe one of the important reason to enhance proliferation, activation and killing ability of CD8+ T cell.Main innovations1. To avoid these limitations connected with peripheral blood and animal model, we choose hepatic tissue as research object, and the research results can reflect the real immune state of liver when the severe hepatitis B is onsetting.2. We observed abnormal expression of KCa3.1 ion channel and CRAC ion channel in CD8+ T cell in the liver of patients with severe hepatitis B. Notably, these ion channels maybe important cause for abnormally increase of cell proliferation, secretion and killing ability of CD8+ T cell.3. It is a new thought and route to investigate the immunopathogenesis of severe hepatitis B through ion channel of T lymphocyte.
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