Proteomic analysis of autoantibodies in patients with hepatocellular carcinoma
Proteomic analysis of autoantibodies in patients with hepatocellular carcinoma
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DOI：
10.1002/pmic.200500346
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发表时间：
2006-07-01
期刊：
影响因子：
3.4
通讯作者：
Nakamura, Kazuyuki
中科院分区：
文献类型：
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作者：
Takashima, Motonari;Kuramitsu, Yasuhiro;Nakamura, Kazuyuki
为了检测可作为肝细胞癌（HCC）诊断标志物的自身抗体，我们综合分析了从HCC患者获得的血清自身抗体，这些抗体对肿瘤组织中蛋白质具有免疫反应性。研究中使用了15个配对的HCC组织和相应的非肿瘤肝组织样本以及5个正常肝组织样本。结合蛋白质组学和SEREX（重组cDNA表达文库的血清学分析）技术。组织蛋白用2-DE分离，转移到PVDF膜上，用自体血清免疫印迹。通过比较每种免疫印迹模式，我们在肿瘤组织中发现了四个免疫反应点，其染色强度高于相应的非肿瘤组织和正常肝组织。采用LC-MS/MS对2-DE凝胶上的匹配蛋白进行鉴定。这些免疫反应蛋白为热休克70 kDa蛋白1 （HSP70）、甘油醛3-磷酸脱氢酶、过氧化物还蛋白和锰超氧化物歧化酶（Mn-SOD）。在HCC血清中，针对这些蛋白的自身抗体的发生率分别为7/15（46.7%）、5/15（33.3%）、5/15（33.3%）和6/15(40.0%)，而对照血清中针对这些蛋白的自身抗体的发生率为2/20（10.0%）、7/20（35.0%）、0/20（0.0%）和2/20（10.0%）。使用市售纯化蛋白进行免疫印迹分析以确认自身抗体的特异性。通过统计分析，肝癌血清中FISP70、过氧化物还氧蛋白、Mn-SOD自身抗体表现出明显的高频免疫反应。这三种抗体被认为是HCC患者特异性抗体，可能是HCC的候选诊断生物标志物。
To detect autoantibodies that could be diagnostic markers for hepatocellular carcinoma (HCC), we analyzed serum autoantibodies comprehensively that showed immunoreactivity to proteins in tumor tissue obtained from patients with HCC. Fifteen paired samples of HCC tissue and corresponding nontumorous liver tissue as well as five normal liver tissue samples were used in the study. A combination of proteomics and SEREX (serologic analysis of recombinant cDNA expression libraries) technique was used. Tissue proteins were separated by 2-DE, transferred onto PVDF membranes, and immunoblotted with autologous sera. By comparing each immunoblot pattern, we identified four immunoreactive spots with stronger staining intensity in tumorous tissues than in corresponding nontumorous tissues and in normal liver tissues. Matched proteins on 2-DE gels were identified by LC-MS/MS. These immunoreactive proteins were heat shock 70 kDa protein 1 (HSP70), glyceraldehyde 3-phosphate dehydrogenase, peroxiredoxin, and manganese superoxide dismutase (Mn-SOD). In HCC sera, occurrences of autoantibodies against these proteins were 7/15 (46.7%), 5/15 (33.3%), 5/15 (33.3%), and 6/15 (40.0%), respectively, whereas 2/20 (10.0%), 7/20 (35.0%), 0/20 (0.0%), and 2/20 (10.0%) were in control sera. Immunoblot analysis using commercially available purified proteins was performed to confirm the specificity of autoantibodies. By statistical analysis, autoantibodies against FISP70, peroxiredoxin, and Mn-SOD showed significantly high-frequency immunoreaction in HCC sera. The three antibodies were considered patient-specific antibodies in HCC and may be candidate diagnostic biomarkers for HCC.