中文摘要：

目的鉴定肝癌患者血清差异表达蛋白并在肝癌细胞和组织中验证肝癌潜在标志物Clusterin的表达水平。方法运用同位素标记相对和绝对定量（iTRAQ）结合LC—MALDI-TOF／TOF串联质谱技术，分析20例肝癌患者与20例健康对照的血清中差异蛋白的表达水平，用实时定量RT_PCR检测潜在标志物Clusterin在肝癌细胞和30对肝癌和癌旁组织中mRNA表达水平;Western blot检测Clusterin在肝癌细胞中的表达水平。肝癌细胞株与正常肝细胞株均数的比较采用两样本t检验，肝癌组织和癌旁组织均数的比较采用配对t检验。结果利用iTRAQ结合质谱分析共发现显著差异表达蛋白51个，其中Clusterin在肝癌患者血清中表达下调IRT-PCR结果显示，Clusterin在正常肝细胞中的mRNA表达水平是肝癌细胞中表达水平的20倍（t=-3．482，P=0．0017），癌旁组织中的mRNA表达水平是肝癌组织中表达水平的2．38倍（t=-2．840，P=0．0086）;Western blot检测Clusterin在肝癌细胞和正常肝细胞中的吸光度值分别为8．06和27．81（t=-3．256，P=0．0015），在肝癌细胞中的表达明显降低。结论Clusterin在肝癌患者血清、肝癌细胞和组织中的mRNA表达水平和在肝癌细胞中表达水平均为下调，其与肝癌的关系值得进一步研究。

英文摘要：

Objective To determine the differentially expressed serum proteins in patients with hepatoma carcinoma and identify a putative diagnostic marker. Method The isobaric tags for relative and absolute quantitation （iTRAQ） labeling method and LC-MALDI-TOF/TOF MS detection method were used to quantify serum proteins in hepatocellular carcinoma patients （n = 20） and healthy individuals （n = 20）. Real- time reverse transcription-polymerase chain reaction was used to verify the differentially expressed proteins by analyzing the corresponding mRNA expression levels in the hepatic carcinoma and healthy hepatocyte samples, as well as in 30 pairs of patient-matched hepatic carcinoma and adjacent normal tissue samples. Western blot analysis was used to verify the protein expression in hepatic carcinoma cells. Results Fifty-one proteins were significantly differentially expressed between the hepatic carcinoma group and healthy controls. The iTRAQ protein profile showed that the serum level of clusterin was significantly lower in hepatoma carcinoma patients. The mRNA level of clusterin was 20-fold lower in hepatic carcinoma cells than in healthy hepatocytes, and was 2.38-fold lower in hepatoma tissues than that in adjacent normal tissues. The clusterin protein levels were significantly lower in hepatic carcinoma cells （8.06 vs normal hepatocytes： 27.81;P〈 0.01）. Conclusions The serum expresion of clusterin is significantly decreased in both serum and tissues of hepatic carcinoma patients. The relationship between hepatic carcinoma and clusterin should be evaluated in future studies.