| [Background and Aims]After HBV infected hepatocyte, the viral antigen was degraded to oligopeptide by antigen presenting cells (APC), then combined with HLA-1 molecular inside the hepatocyte to form complex and expressed on the surface of cells, which was called T cell epitope. Cytotoxic lymphocytes (CTL) adhered to HLA-1 molecular through CD8+ molecular in the cell surface, and TCR recognized above T cell epitope. Such kind CTL was called HBV antigens specific cytotoxic lymphocytes. Recent studies showed that response of HBV antigens specific cytotoxic lymphocytes has important significance in HBV infection. HBV antigens specific cytotoxic lymphocytes secreted INF- T to clear HBV without hepatocyte damage. The subsequent hepatocyte damage maybe relate with the infiltration or apotosis of non-specific immune active cell in liver. Further study showed that, the difference of HBV specific response function closely related with the different clinical prognosis of HBV patients. Specific cytotoxic lymphocytes responsed strongly in acute hepatitis, which may relate with the quick clearance of HBV and mild liver damage. In chronic hepatitis, weak response of specific cytotoxic lymphocytes may relate with infiltration of inflammatory cells. Another study showed that stimulating the specific cytotoxic lymphocytes maybe the efficient way for chronic hepatitis to clear the virus and gain recovery. In this paper, we selected a new lab technology called human histocompatibility leukocyte antigen (HLA)-peptide tetramers flow cytometry to study HBV specific cytotoxic lymphocytes in severe hepatitis B, in order to providesome new ideas and technology for further study and prevention of hepatitis B. [Material and Methods]1. Patients and controls: 34 cases of chronic hepatitis B patients were divided into two groups by histology technology. The former 19 cases HLA-A2+ group including 11 cases male and 8 female, from 16 to 54 years old, average 32 years old, and the remaining 15 cases HLA-A2" served as control group including 10 cases male and 5 female, from 28 to 49 years old, average 34 years. 25 cases of HBsAg positive carrier included 14 cases HLA-A2+ (7 cases males and 7 cases female, average 36 years old), 11 cases HLA-A2" as control (5 cases males and 6 cases female, average 34 years old). 10 cases of acute hepatitis included 6 cases HLA-A2+(5 cases males and 1 cases female, average 42 years old), 4 cases HLA-A2" as control (3 cases males and 1 cases female, average 35 years old). 42 cases of HLA-A2+ severe hepatitis were divided into two groups. One was the group of 20 cases of acute severe hepatitis (12 cases males and 8 cases female, average 30 years old), the other was the group of 22 cases of chronic severe hepatitis (15 cases males and 5 cases female, average 45 years old). All these patients were admitted in our hospital from Feb.2002 to Aug. 2003. 15 cases of HLA-A2+healthy as control with markers as HAV, HBV, HCV, HDV, HEV and HIV negative.2.Methods:Separation of peripheral blood mononuclear cells (PBMC): Whole blood samples were collected from patient's vein in morning. PMSB were separated from lymphocyte separations.HLA typing: Screening for the HLA-A2 haplotype was performed by flow cytometry, then confirmed by sequence specific primer polymerase chain reaction (SSP-PCR).Tetramer flow cytometry to detect specific cytotoxic lymphocytes in PBMC: HBV core 18-27, polymerase 575-583 and envelope 335-343 specific CD8+T cells were detected by flow cytometry using PE staining tetramer and FITC staining anti-CD8+, HCV C region 178-187 epitope tetramer served as control. The number of CD8+ and tetramer double positive cells was counted as specific CD8+ cells among 50000 CD8+ cells.INF- T , TNF- a , IL-4 and IL-10 ELISPOT assays: According to the introduction of kit, specific antibody to the cytokine was coated in microplate wells, then PBMCs co-stimulated with HBV corel8-27 peptide and rHBcAg for 36 hours and the second antibody conjugated to Alkaline Phosphatase were added and incubated f... |
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