Takashima Motonari, Kuramitsu Yasuhiro, Yokoyama Yuichiro, Iizuka Norio, Harada Toshio, Fujimoto Masanori, Sakaida Isao, Okita Kiwamu, Oka Masaaki, Nakamura Kazuyuki
Department of Surgery II, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan.
Proteomics. 2006 Jul;6(13):3894-900. doi: 10.1002/pmic.200500346.
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 HSP70, 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.
为了检测可作为肝细胞癌（HCC）诊断标志物的自身抗体，我们全面分析了对HCC患者肿瘤组织中的蛋白质具有免疫反应性的血清自身抗体。本研究使用了15对HCC组织和相应的非肿瘤肝组织样本以及5份正常肝组织样本。采用了蛋白质组学和SEREX（重组cDNA表达文库血清学分析）技术相结合的方法。组织蛋白通过双向电泳（2-DE）分离，转移到聚偏二氟乙烯（PVDF）膜上，并用自体血清进行免疫印迹。通过比较每种免疫印迹模式，我们在肿瘤组织中鉴定出4个免疫反应斑点，其染色强度比相应的非肿瘤组织和正常肝组织更强。通过液相色谱-串联质谱（LC-MS/MS）鉴定了2-DE凝胶上匹配的蛋白质。这些免疫反应性蛋白质分别是热休克70 kDa蛋白1（HSP70）、甘油醛-3-磷酸脱氢酶、过氧化物酶和锰超氧化物歧化酶（Mn-SOD）。在HCC血清中，针对这些蛋白质的自身抗体出现率分别为7/15（46.7%）、5/15（33.3%）、5/15（33.3%）和6/15（40.0%），而在对照血清中分别为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，针对HSP70、过氧化物酶和Mn-SOD的自身抗体在HCC血清中显示出显著高频的免疫反应。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。
