HBV相关性肝细胞癌及肝硬化患者外周血单个核细胞质谱分析

Mass spectrometric analysis of peripheral blood mononuclear cells in patients with hepatitis B virus associated hepatocellular carcinoma or liver cirrhosis

目的:建立HBV相关性肝细胞癌(HCC)及肝硬化(LC)患者外周血单个核细胞(PBMC)的双向凝胶电泳图谱,并识别鉴定其差异表达的蛋白质.方法:利用固相pH梯度双向凝胶电泳分离HBV相关性HCC(n=16)及LC患者(n=12)PBMC的总蛋白质,凝胶经蓝银显色后,PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)得到相应的肽质指纹图谱,然后搜索数据库鉴定部分差异蛋白质点.结果:得到了分辨率较高、重复性较好的HBV相关性HCC及LC患者PBMC双向凝胶电泳图谱,HBV相关性HCC及LC患者PBMC凝胶的平均蛋白质点数分别为1186±43及1013±41;组内的平均匹配率分别为91.6%,90.2%.对27个差异蛋白质点进行了质谱分析,鉴定出16个蛋白质,在HBV相关性HCC患者PBMC中表达明显增强的有热休克蛋白质(HSP)27、ras癌基因家族(RAB14)、肌动蛋白、α1-抗胰蛋白及RNA结合蛋白调节亚基等13个蛋白点;而PDX6、HSPA8及锰超氧化物歧化酶(MnSOD)在HCC患者PBMC中低表达.这些差异蛋白质点包括蛋白质合成与分解、分子伴侣、解毒和DAN修复相关蛋白质、三大代谢相关酶类、细胞结构相关蛋白质以及信号传导相关蛋白质六类.结论:建立了分辨率高且重复性较好的HBV相关性HCC及LC患者PBMC双向凝胶电泳图谱,并识别鉴定出了一些与HBV相关性HCC癌变相关的差异表达的蛋白质.

AIM： To establish two-dimensional electrophoresis （2-DE） maps of peripheral blood mononuclear cells （PBMC） in patients with hepatitis B virus （HBV） associated hepatocellular carcinoma （HCC） or liver cirrhosis （LC）, and identify the differentially expressed proteins. METHODS： The total proteins from PBMC in patients with HBV associated HCC （n = 16） or LC （n = 12） were seperated by means of immobilized pH gradient-based 2-DE. The differentially expressed proteins were screened using PDQuest analysis software, and then identified by peptide mass fingerprint （PMF） based on matrixssisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： A well-resolved and reproducible 2-DE pattern of PBMC was obtained from patients with HBV associated HCC or LC. For HCC, the mean number of protein spots was 1186 ± 43, with an average matching rate of 91.6%, and for LC, the mean number of protein spots was 1013±41, with an average matching rate of 90.2%. Twenty-seven differential proteins were tested by MALDI-TOF- MS, and 16 were identified. Of the 16 proteins, 13 were up-regulated in patients with HCC, including heat-shock protein 27, member RAS oncogene family （RAB14）, actin, alpha 1 antitrypsin, RNAbinding protein regulatory subunit and so on. By contrast, the levels of proteins such as PDX6, HSPA8 and MnSOD were significantly reduced in HCC patients. Some of these proteins participated protein synthesis and degradation, or chaperone, or protection and detoxification, or signal transduction, while some of them were involved in metabolism or cytoskeleton construction. CONCLUSION： A well-resolved and reproducible 2-DE pattern of PBMC from patients with HBV related HCC or LC is established, and sixteen differentially expressed proteins were characterized.