Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye
Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye
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DOI：
10.1002/pmic.200401004
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发表时间：
2005-04-01
期刊：
影响因子：
3.4
通讯作者：
Hirohashi, S
中科院分区：
文献类型：
--
作者：
Fujii, K;Kondo, T;Hirohashi, S
为了确定与肝细胞癌 (HCC) 癌变相关的蛋白质组改变，我们将九个 HCC 细胞系的蛋白质表达谱与从五个个体建立的原代培养肝细胞的蛋白质表达谱进行了比较。通过二维差异凝胶电泳进行差异蛋白质组学研究，其中蛋白质样品用不同的荧光染料标记，并根据其等电点和分子量进行分离。为了标记蛋白质样品，我们使用了新开发的高灵敏度荧光染料，它与蛋白质的所有还原半胱氨酸残基发生反应。基于1238个蛋白点强度的主成分分析表明，HCC细胞和正常肝细胞具有不同的蛋白质组谱。采用Wilcoxon检验确定HCC细胞与正常肝细胞中强度差异调节的蛋白点，并采用质谱分析来鉴定与该点相对应的蛋白质。鉴定出的蛋白质涉及细胞周期调节、与肿瘤抑制基因产物的结合、脂肪酸结合和翻译调节。特异性抗体的蛋白质印迹显示，与非癌组织相比，HCC 组织中 PCNA、EB1 和 E-FABP 过度表达，EB1 和 E-FABP 的异常调节此前并未涉及 HCC 的发生。
To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238 protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues, Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC.