【作者】 刘惠；

【导师】 邵宗鸿； 付蓉；

【作者基本信息】 天津医科大学 ， 内科学， 2010， 博士

【摘要】 目的：检测免疫相关性全血细胞减少(immuno-related pancytopenia, IRP)患者自身抗体(IgG／IgM)在骨髓红系造血细胞及其他造血细胞膜上的作用靶点,探索IRP患者造血细胞膜上可能的靶抗原成分,为进一步纯化和克隆IRP患者自身抗原提供依据。方法：研究对象为66例初治IRP患者及28名正常对照。应用流式细胞术(FACS)检测骨髓红系造血细胞膜表面自身抗体与促红细胞生成素受体(EPOR)的数量,观察有核红细胞自身抗体阳性组与自身抗体阴性组IRP患者EPOR的表达水平；RT-PCR检测EPOR mRNA的表达水平；FACS分选骨髓红系造血细胞后,免疫印迹法检测Stat5及P-Stat5蛋白表达水平以观察EPO／EPOR信号通路是否受抑；利用甘氨酸缓冲液洗脱骨髓红系造血细胞膜上结合的自身抗体,重新标记后再次检测EPOR的表达水平；FACS检测骨髓造血细胞膜抗体,提取细胞膜蛋白,免疫印迹法检测IRP患者骨髓上清液中自身抗体IgG的阳性率,进而应用聚丙烯酰胺凝胶电泳结合免疫印迹法分离和鉴定IRP患者自身抗体IgG在细胞膜上的靶抗原,并与正常对照及病例对照组进行对比；选取目标蛋白条带进行高压液相串联质谱(LC-MS／MS)分析以确定靶抗原成分。结果：1.(1)有核红细胞自身抗体阳性组IRP患者有核红细胞膜EPOR表达量为(1.59±0.87)%,明显低于自身抗体阴性组(4.58±4.09)%(P<0.01),后者则明显高于正常对照组(2.27±1.76)%(P<0.05)；IRP患者有核红细胞膜上EPOR表达水平与自身抗体呈显著负相关(r=-0.543,p=0.000),EPOR(Y)与自身抗体(X)的直线回归方程为Y=0.040-0.335X；有核红细胞自身抗体阳性组网织红细胞百分率为(1.55±0.64)%,显著低于自身抗体阴性组(2.41±1.42)%(P<0.05)；IRP患者有核红细胞膜上EPOR表达水平与其网织红细胞百分率呈显著正相关(r=0.346,p=0.029)；(2)有核红细胞自身抗体阳性组EPOR mRNA表达水平为(0.685±0.136),明显高于自身抗体阴性组(0.554±0.116)(P<0.01)和正常对照组(0.580±0.119)(P<0.05)；(3)有核红细胞自身抗体阳性组Stat5蛋白表达水平为(1.45±0.94),明显高于正常对照组(0.54±0.36)(P<0.05),而自身抗体阴性组Stat5蛋白表达水平为(0.75+0.69),与正常对照组无统计学差异(P>0.05)；自身抗体阳性组P-Stat5蛋白表达水平为(0.42±0.18),明显低于正常对照组(0.85±0.38)(P<0.05),而自身抗体阴性组P-Stat5蛋白表达水平为(0.70±0.18),与正常对照组无统计学差异(P>0.05)；(4)应用甘氨酸洗脱液洗脱抗体后自身抗体数量明显减少,而EPOR表达水平明显增高,提示部分自身抗体洗脱前封闭EPOR。2.IRP组骨髓上清液中IgG的阳性率为75%(15／20),明显高于病例对照组(0%)和正常对照组(10%,1／10)(均P<0.01)。在20例初治IRP患者中,其骨髓造血细胞膜上共有五种蛋白成分可被自身抗体IgG识别,其相对分子量分别为25-30kDa、47.5kDa、60-65 kDa、73 kDa和83 kDa,其阳性率分别为35%(7／20)、10%(2／20)、50%(10／20)、20%(4／20)、10%(2／20)。6例再生障碍性贫血(AA)患者及4例骨髓增生异常综合征(MDS)患者经免疫印迹法鉴定后均未发现细胞膜上有蛋白被自身抗体IgG识别。正常对照组可见相对分子量为90kDa和150kDa的蛋白被识别,其阳性率均为10%(1／10)。选取25kDa、30kDa和47.5 kDa蛋白条带进行高压液相串联质谱分析,发现G蛋白偶联受体156变异体及人红细胞带3蛋白胞内区域结晶体,P链两种膜蛋白成分。结论：部分IRP患者自身抗体可作用于骨髓红系造血细胞膜上的EPOR,封闭EPOR信号转导,导致红系较早阶段增殖和分化受到抑制。EPOR为自身抗体作用的靶点之一；IRP患者骨髓上清液中存在自身抗体IgG,可作用于骨髓细胞膜上多个靶抗原成分,质谱鉴定结果含G蛋白偶联受体156变异体及人红细胞带3蛋白胞内区域结晶体,P链两种膜蛋白成分。更多还原

【Abstract】 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.更多还原