摘要目的 筛查潜在的小分子量低丰度的HCC血清相关候选蛋白,为肝癌复杂病理机制的全面阐明、理想早期诊断标志物的发现提供新思路.方法 分别收集81例伴有HBV感染和肝硬化背景的HCC患者血清,43例慢性乙型肝炎和36例肝硬化患者血清,采用弱阳离子交换蛋白芯片(WCX2)介质结合SELDI-TOF-MS对患者血清蛋白谱进行检测,并通过Biomarker Wizard软件对生成的蛋白指纹峰分别进行差异归类比较,然后采用消减差异分析确定HCC特异相关候选蛋白.标本检测前,对芯片检测系统的重复性进行分析.结果 同一标本同一芯片内强度CV为17.46%,m/z平均CV为0.024%,不同芯片间强度CV为17.74%,m/z平均CV为0.024%.将信噪比大于5,且超过20%标本具有的蛋白峰认定为有意义峰,在2 000～30 000 m/z范围内,共有128个蛋白指纹峰被确认.HCC与肝硬化患者血清蛋白指纹峰的差异比较分析显示,87个差异蛋白指纹峰有统计学意义(P＜0.05),其中强度差异在2倍以上的蛋白指纹峰有45个,在HCC患者血清中2倍上调的15个,2倍下调的30个.从HCC与慢性乙型肝炎血清蛋白指纹峰的差异分析发现,差异有统计学意义的蛋白指纹峰52个(P＜0.05),其中强度相差2倍以上的蛋白指纹峰有9个,HCC患者血清中2倍上调的2个,2倍下调的7个.从肝硬化与慢性乙型肝炎患者血清蛋白指纹峰的差异比较分析发现,差异有统计学意义的蛋白指纹峰79个(P＜0.05),其中强度相差2倍以上的蛋白指纹峰有28个,在肝硬化患者血清中2倍上调的17个,慢性乙型肝炎患者血清中2倍上调的11个.经消减差异分析筛查,确定在HCC患者血清下调表达的5个公共差异蛋白(2 870、3 941、2 688、3 165、5 483 m/z)及在肝硬化和HCC血清均上调表达的2个公共差异蛋白(3 588、2 017 m/z),但蛋白2 017 m/z在HCC与健康人比较的本所前期工作中的差异无统计学意义(P＞0.05).结论 由慢性乙型肝炎和肝硬化背景产生的非特异分泌蛋白的干扰,经消减分析被部分消除,获得的6个血清公共差异蛋白(2 870、3 941、2 688、3 165、5 483、3 588 m/z)在反映HCC病理特征方面具有明显的特异性,且有望成为HCC诊断中新的候选标志物.

abstractsObjective To screen potential serum HCC associated proteins with low molecular weight and low abundance for better understanding the pathological mechanism of HCC and discovering new biomarkers.Methods All serum samples were collected from 81 HBV-related HCC patients,43 chronic hepatitis B patients and 36 cirrhosis patients.Serum protein fingerprint profiles were first generated by selected WCX2 protein chip integrating with SELDI-TOF-MS,and then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard.Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra was performed.Some protein peaks with significant difference among HCC,cirrhosis and chronic hepatitis B groups were found.The reproducibility of the SELDI system was assessed before serum protein fingerprint profiles analysis.Results The intra-and inter-assay CV for intensity and m/z in this SELDI system were 17.46% and 0.024%,and 17.74% and 0.024% respectively.Total 128 protein fingerprint peaks between 2 000 to 30 000 Da were identified under the condition of signal to noise＞5 and minimum threshold for cluster＞20%.Eighty-seven proteins were found to significantly expressed between HCC and cirrhosis groups(P＜0.05).Of the above differential proteins,forty-five proteins had changes greater than two fold,including 15 up-regulated proteins and 30 downregulated proteins in HCC sera.Between HCC and chronic hepatitis B groups,nine of fifty-two differential proteins(P＜0.05) had intensities of more than two folds,including 2 up-regulated proteins and 7 downregulated proteins in HCC sera.Between cirrhosis and chronic hepatitis B groups,twenty-eight of seventynine significantly differential proteins(P＜0.05) changed greater than two folds in intensity,including 17 up-regulated proteins in cirrhosis seru and 11 down-regulated proteins in chronic hepatitis B sera.Analysis of above leading differential proteins among three diseases using subtraction difference mode,the 5 common down-regulated proteins 2 870,3 941,2 688,3 165 and 5 483 m/z in HCC sera and 2 common up-regulated proteins 3 588 and 2 017 m/z in cirrhosis and HCC sera were screened.But no statistic difference in the level of protein 2 017m/z was found between HCC group and normal group inour previous study.Conclusion Because the interference of unspecific proteins from hepatitis B and cirrhosis could be eliminated partly in HCC sera through subtraction difference analysis,these 6 common differential proteins (2 870,3 941,2 688,3 165,5 483,3 588 m/z)have obvious advantages of increased specificity for evaluating the pathological state of HCC and might become promising candidate biomarkers in the diagnosis of HCC.