狼疮肾炎中新发现的抗肾小球系膜细胞抗体靶抗原的鉴定

Characterization of target antigens of the novel anti-mesangial cell antibodies in sera from patients with lupus nephritis

目的 研究狼疮肾炎血清中新发现的抗肾小球系膜细胞抗体与抗DNA抗体的关系 ,及其靶抗原是否存在于细胞膜上。方法 DNA纤维素柱亲和层析分离狼疮肾炎患者血清中的抗DNA抗体 ,Western印迹法检测祛除抗DNA抗体的血清和亲和层析纯化的抗DNA抗体与系膜细胞靶抗原的结合。从体外培养的人系膜细胞系分离细胞膜并提取膜蛋白 ,以已知抗肾小球系膜细胞抗体阳性血清为探针应用Western印迹法鉴定膜蛋白中的靶抗原。结果 系膜细胞中相对分子质量为74 0 0 0、36 0 0 0的蛋白可以被祛除抗DNA抗体的血清识别。而相对分子质量为 6 30 0 0、910 0 0、12 5 0 0 0的蛋白被亲和层析的抗DNA抗体识别。系膜细胞膜上至少相对分子质量为 10 10 0 0、910 0 0、74 0 0 0和 310 0 0的蛋白可以被狼疮肾炎患者的血清识别。结论 部分抗肾小球系膜细胞抗体与抗DNA抗体无关。人系膜细胞系的膜蛋白中存在狼疮肾炎血清可识别的抗原物质 ,提示抗DNA抗体以外的直接针对肾小球系膜细胞的自身抗体在狼疮性肾炎的发病机制中可能起重要作用。

Objective Novel anti-mesangial cell antibodies have been identified in sera of majority patients with lupus nephritis in our previous study. The aims of current study were to investigate the association of anti-mesangial cell antibodies and anti-DNA antibodies, and whether the target antigens were on mesangial cell membrane. Methods Affinity parified anti-DNA antibodies and sera with depletion of anti-DNA antibodies from three patients with lupus nephritis were used in Western-blot analysis to identify antigens in culture human glomerular mesangial cell line. Cell membrane from in vitro cultured human mesagial cell line was separated by gradient centrifugation technique,and the target antigens were identified by Western-blot analysis using 1% Triton X-100 solublized membrane protein as antigens and four known anti-mesangial cell antibody positive sera from patients with lupus nephritis as probes. Results Soluble proteins from in vitro cultured human mesangial cells could be blotted by both affinity purified anti-DNA antibodies (molecular weight at 63 000, 91 000 and 125 000) and sera with depletion of anti-DNA antibodies (molecular weight at 74 000 and 36 000). Proteins with molecular weight at 101 000, 91 000, 74 000 and 31 000 in mesangial cell membrane could be blotted by the four known anti-mesangial cell antibody positive sera. Conclusion Some anti-mesangial cell antibodies were not associated with anti-DNA antibodies and anti-mesiangial cell antibodies could direct interact with antigens located on mesangial cell membrane. Anti-mesangial cell antibodies may play an important role in the pathogenesis of lupus nephritis.