Study On Anti-liver Antigens In Patients With Autoimmune Liver Diseases

Autoimmune liver diseases can be divided into three subtypes: autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis. This kind of diseases is usually characterized by a chronic inflammatory disease process with lymphocytic infiltration, hypergammaglobulinemia and characteristic autoantibodies. The reason for this inflammation is not certain, but it is associated with an abnormality of the body's immune system and is often related to the production of antibodies that can be detected by varied tests. The reports have shown that early detecting, early diagnosis and early treatment has make easy to control the disease, to improve the patients living condition. These autoantibodies are often valuable diagnostic markers and sometimes act as hallmark for autoimmune liver diseases.ANA with/without SMA are the principal serologic markers of type 1 AIH. Detection of these autoantibodies is widely used, but ANA and SMA are not disease-specific, furthermore these autoantibodies are not associated with the disease processes. Anti-LKMl typically occurs in the type 2 AIH. Anti-SLA has been promulgated as a highly specific marker of a type 3 AIH. Detecting and investigating autoantiboies are useful in identifying and classifying liver disease clinically, and also provide important non-traumatic diagnostic markers for AIH. So finding new liver-associated autoantibodies is helpful not only for diagnosis but also for pathogenesis research in autoimmune liver diseases. This study was divided into three parts.Part 1. Expressing and purifying soluble liver antigen and CYP2D6SLA cDNA and CYP2D6 cDNA obtained from human liver tissue poly (A) +RNA by RT-PCR. The cDNAs were inserted into fusion expression vector pQE-30site of BamH I and Hind III, SLA and CYP2D6 fusion proteins were high expressed in E.Coli Ml5. After identified by SDS-PAGE and Westemblotting, SLA and CYP2D6 were purified by Ni-NTA affinity chromatography. SDS-PAGE analysis showed that there were very strong stained bands at about 47.5kD and 50kD, and the result of western blotting showed that the products specially bind to anti-SLA or anti-CYP2D6 autoantibodies. After purified, the SDS-PAGE analysis showed there was one band corresponding to SLA or CYP2D6 purified. To success on expression and purification of SLA and CYP2D6 not only is helpful to the diagnosis but also provide useful material for the research of pathogenesis of autoimmune hepatitis.Part 2. To detect the autoantibodies in autoimmune liver disease patients and analysis the positive ratioDetecting a group of autoantibodies in 178 autoimmune liver disease patients and 200 normal adults with the use of indirect immunofluorescence assay and immunoblot assay. The patients were divided into 3 groups: AIH, PBC and unknown cause liver injury and then we analyzed the positive ratio of autoantibodies in each group. The AIH group was divided into 3 subtypes according to the different patterns of the autoantibodies as I (75%), II (8.3%), III (16.7%). In the PBC group, the patients are all AMA positive, the positive rate of M2 is 95.5%. The autoantibodies appear in 80.0% unknown cause liver injury group in which there are also 22 patients found none autoantibodies and 5 cases of other patients presented unknown autoantibodies. One of the unknown autoantibodies, found in the patient A, appeared liver specific. The immunofluorescent pattern for the unknown autoantibody showed that : (1) positivity on hepatocyte cytoplasm but none in kidney; (2)stronger fluorescence in the central vein zone and decreased from central to peripheral. In Westemblotting test, the positive band was found, which related molecular mass about 55 to 60kD.Part 3. Screening and analyzing the unknown target autoantigenThe serum of patient A was used as the probe for immunoscreening the A. TriplEx cDNA library. The isolated positive plaque was picked out for secondary andtertiary screening. Then the insert sizes of positive screened plaques were determined by PCR and enzyme digestion, which was 1000bp.