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目的:分析肝癌细胞系HCCLM3的细胞外蛋白降解组,筛选肝癌标记物。方法:凝胶电泳分离HCCLM3细胞外蛋白,线性离子阱-傅立叶变换离子回旋共振质谱(LTQ-FT-MS/MS)对其鉴定,数据库检索结合分子质量分析其降解组。免疫杂交定量骨桥蛋白(OPN)在正常、乙肝、乙肝肝硬化和肝癌4组血清中的表达。结果:HCCLM3细胞外蛋白电泳共测得51个条带,鉴定出1 405种非冗余蛋白,其中474种蛋白发生降解,且分泌蛋白的降解率最高(50%)。肝癌患者血清中OPN 20 kD片段明显高于其余3组(P<0.05),受试者工作特征曲线(ROC)显示AFP对肝癌诊断的曲线下面积(AUC)为0.79,OPN 20 kD片段的AUC为0.68,两者结合后能提高AUC至0.89。结论:凝胶电泳结合质谱技术研究肝癌蛋白降解组有效可行,骨桥蛋白20 kD片段可能对肝癌的诊断具有价值。
OBJECTIVE: To analyze the extracellular protein degradation of HCCLM3 cell line and screen for markers of HCC. METHODS: HCCLM3 extracellular proteins were separated by gel electrophoresis. Their structures were characterized by linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (LTQ-FT-MS / MS). Their databases were searched for molecular mass analysis. Immunohistochemistry Quantitative osteopontin (OPN) in normal, hepatitis B, hepatitis B cirrhosis and liver cancer serum of four groups. Results: A total of 51 bands were detected by extracellular protein electrophoresis in HCCLM3. A total of 1 405 non-redundant proteins were identified, of which 474 proteins were degraded and the secreted protein had the highest degradation rate (50%). The serum OPN 20 kD fragment in patients with HCC was significantly higher than the other three groups (P <0.05). The receiver operating characteristic curve (ROC) showed that the area under the curve (AUC) of AFP for the diagnosis of HCC was 0.79 and that of the OPN 20 kD fragment 0.68, the combination of the two can increase AUC to 0.89. Conclusion: It is feasible and effective to study the protein degradation of liver cancer by gel electrophoresis and mass spectrometry. The 20 kD fragment of osteopontin may be valuable for the diagnosis of liver cancer.