| Objective:To investigate the autoantigens targeted by autoantibodies(IgG/IgM) on the membrane of erythropoietic cells and other bone marrow cells of the patients with immuno-related pancytopenia(IRP),for further purifying and cloning them.Methods:66 newly diagnosed IRP patients and 28 healthy donors as controls were enrolled in this study. EPOR expression on their nucleared erythrocytes were tested by flow cytometry to observe the relationship between EPOR and autoantibodies; EPOR mRNA were tested by RT-PCR to observe the production of EPOR; Stat5 and P-Stat5 proteins in nucleared erythrocytes were measured by Western blot to observe EPO/EPOR signal transduction; EPOR expression on the nucleared erythrocyte membrane were tested again after stripping autoantibodies with glycine buffer to see if autoantibodies had covered EPOR;Autoantibody IgG in bone marrow supernatant was measured by Western blot;The bone marrow cell membrane autoantigens targeted by IgG in IRP were identified from membrane protein extracts by SDS-PAGE, Western blot and liquid chromatography-mass spectrography/mass spectrography.Results:1. (1) EPOR on nucleared erythrocytes of the IRP patients with detected erythrocyte autoantibodies (auto-Ab (+) arm) (1.59±0.87)% was significiantly lower than that of auto-Ab (-) arm (4.58±4.09)%(P<0.01), and the latter was significiantly higher than that of normal controls (2.27±1.76)%(P<0.05); EPOR of IRP patients was inversely correlated with their autoantibodies on erythrocytes (r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(auto-Ab);(2) EPOR mRNA of auto-Ab (+) arm (0.685±0.136) was significiantly higher than those of auto-Ab (-) arm (0.554±0.116) (P<0.01) and normal controls (0.580±0.119) (P<0.05);(3) Stat5 protein of auto-Ab (+) arm (1.45±0.94) was significiantly higher than that of normal controls(0.54±0.36)(P<0.05);While P-Stat5 protein of auto-Ab (+) arm(0.42±0.18) was significiantly lower than that of normal controls(0.85±0.38)(P<0.05); (4) EPOR expression increased significantly after stripping the auto-Ab from nucleared erythrocytes with glycine buffer.2. Autoantibody IgG reacting with bone marrow cell membrane antigens could be found in bone marrow supernatant of IRP patients in a positive rate of 75%(15/20), which was significantly higher than those of aplastic anemia(AA) or myelodysplastic syndrome (MDS) patients(0%) and normal controls(10%) (P<0.01).Autoantibody IgG in IRP could react with several autoantigens with approximate MWs of 25-30kDa,47.5 kDa,60-65 kDa,73 kDa and 83 kDa.Positive retes of individual autoantigens were 35%,10%,50%,20% and 10%,respectively.25kDa,30kDa and 47.5 kDa were identified by liquid chromatography-mass spectrography/mass spectrography as G protein coupled receptor 156 variant and chain P, crystal structure of the cytoplasmic domain of human erythrocyte band-3 protein.Conclusions:The autoantibody of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR was one of autoantigens in IRP. Autoantibody IgG could be found in bone marrow supernatant in IRP.The molecular weights of the autoantigens targeted by IgG were mainly 25-30kDa,47.5kDa,60-65 kDa,73 kDa and 83 kDa. Autoantigens in 25kDa protein were identified as G protein coupled receptor 156 variant and chain P, crystal structure of the cytoplasmic domain of human erythrocyte band-3 protein. |
