【作者】 谈冶雄；

【导师】 王红阳； 吴孟超；

【作者基本信息】 第二军医大学 ， 生物化学与分子生物学， 2002， 博士

【摘要】 肝细胞癌(Hepatocellular Carcinoma，HCC)是最常见的原发性肿瘤，据报道全世界每年HCC死亡人数可能在100万左右。每年新发生26-30万例肝癌中42.5％发生在我国；我国肝癌死亡率在过去的20年间增加了约42％，每年仅我国就有至少30万人死于肝癌。 目前肝细胞癌临床诊疗的两大难点是早期诊断难和极高的复发转移率，相关研究热点是筛选新的标志分子和解析肝癌复发转移的相关基因及其分子机制。虽然目前已知与肝癌的转移复发相关的分子有很多，如：癌基因(c-myc、c-fos、N-ras)、肿瘤抑制基因(p53、Ki-1、Rb)、肝细胞生长因子、转化生长因子、胰岛素样生长因子、内皮细胞钙粘蛋白、内皮细胞选择素、细胞间粘附分子-1、整合素、CD44、层粘蛋白受体、促细胞移动因子-Rho和Rho相关的蛋白激酶、基质降解酶、促血管生成因子等，但这些研究大多为与肝癌复发转移关联的非系统性研究，至今仍不能对肝癌的复发转移的机制有相对全面和系统的了解。目前的共识是：肝癌的复发转移是多个分子共同作用，不同的信号转导途径的异常与相互影响的结果。寻找更多这样的分子和阐明作用机制需要在整体水平上分析肝癌细胞内的动态变化。此外，蛋白是基因功能的执行者，多数肿瘤是多种功能蛋白全局性的紊乱，解析其中的规律是单个基因的研究难以实现的。近几年，以双向电泳、质谱分析为主的蛋白质组学的方法和手段为肿瘤研究提供了良好的技术平台。 本课题中，我们与中科院生物化学和细胞生物学研究所夏其昌教授课题组合作，对复旦大学医学院肝癌研究所建立的具有高低侵袭转移能力的两个肝癌细胞系进行了比较蛋白质组分析。经质谱鉴定最明显(有或无)的差异位点为细胞角蛋白19(cytokeratin 19，CK19)。本结果及后期他们进行的转移能力逐步提高的细胞系的CK19表达分析的结果提示CK19表达可能与肝癌细胞的转移有关。 进一步，我们对东方肝胆外科医院收治的肝细胞癌病人的肝癌组织样本进行免疫组化检测。发现在60例肝细胞癌中有8例的部分癌细胞中有CK19表达；更有意义的是，CK19表达情况与肿瘤有无子灶和癌栓等表示肿瘤转移能力的指标密切相关，表明CK19的表达可以作为肝癌细胞高转 －＿－谈冶雄“细胞角蛋白19与肝细胞癌转移相关性的初步研究” 导师：王红阳 教授 吴盂超 院士… 移能力的标志分子。－－应用 CK19单克隆抗体，Western blot检测三种肝癌细胞系：HepG上。 －－＿SK－Hepl和 Hub刁以及胎肝细胞系 W趾68、正常肝细胞系 Changliver的 CK19表达情况。结果显示在HepG上和 SK0epl两种肝癌细胞系中也有－－CK19的表达，证明CK19并非胆管细胞特有，肝细胞在病理情况下可表达 CK。 f 为深入研究 CK与肝细胞癌细胞转移能力的关系，我们由乳腺癌 ·细胞系MCF刁克隆了CK19全长CDNA，构建了pCDNA3－CK19表达载体， 建立了稳定表达CK19的肝癌细胞系Hub7。Boyden Chamber模型的细胞 －迁移实验显示稳定转染CK19的Hllh7迁移能力较对照组明显升高，从而＿＿表明 CK可提高肝癌细胞的转移潜能。 对肿瘤的比较蛋白质组学研究以前多在细胞系中进行，但体外建立。－的肿瘤细胞株与体内肿瘤细胞的生物学性质还是有相当的差别。另外，肿 瘤的个体异质性很高，不同致病原因诱发的肿瘤其蛋白表达行为更可能会－＿有关键性的差别。因此，只有对临床肿瘤组织进行直接的比较蛋白质组学 研究，才能更真实地阐明体内肿瘤的生物学特征，更高效地找到新的标志 分子，提出新的干预手段。－本课题为开展肝癌的比较蛋白质祖研究，在国内首先建立了肝癌组织 的双向电泳分析技术。比较了不同样品处理方法（机械裂解和液氮碾磨法）＿－和取样方法（Percoll梯度分离直接取样、P6rcoll梯度分离后原代培养）对 蛋白表达的双向电泳图谱的影响，为后续肝癌组织的比较蛋白质组研究奠 定了基础。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is among the most frequent cancers worldwide, especially in less developed and developing countries including China.Mechanisms for carcinogenesis and development of HCC is still far from clear. Current research is mainly focused on screening of novel tumor-specific markers and elucidating the molecules and mechanisms responsible for relapse and metastasis of HCC.Although a lot of molecules such as oncogenes, growth factors, adhesion molecules, have been shown to be related to relapse and metastasis of HCC, how they work and interact is poorly understood. Concerning the high heterogeneity and complex processes of HCC, It is urgent now to clarify the global > comprehensive change of signal transduction pathways during carcinogenesis, development, relapse and metastasis. The ever-improving proteomics technologies, mainly 2 dimensional electrophoresis and mass chromatography, may become an ideal platform for it.In this paper, co-operating with Prof. Xia from Shanghai Institute of Biological Chemistry, We screened out a differentially-expressed protein dot on 2D profile between two HCC cell lines (constructed by Medical College of Shanghai Fudan University )different only in metastatic capacity and identified that it is cytokeratin 19(CK19)with MS analysis, which implies the possible role of CK19 in metastasis60 paraffin-embedded HCC samples were screened for CK19 expression. 8 samples showed CK19-positive signals and their expressions were closely related to metastatic clinical indices. Moreover, CK19 is also expressed in two HCC cell lines: HepG-2 and SK-Hepl.To further elucidate the relation between CK19 expression and metastasis of HCC, CK19 cDNA was cloned from breast cancer cell line MCF-7 andpcDNA3-CK19 expression plasmid was constructed.Then Huh-7(HCC cell line without endogenous CK19 expression) was transfected with pcDNA3-CK19 and Huh-7 stably expressing CK19 was established. The migration capacity of this CK19-over-expressing Huh-7 was evaluated through Boyden Chamber test, which showed that CK19 increases dramatically the migration of Huh-7.The past researches of comparative proteomics on HCC are restricted to cell line-based study. But the state and inner pathways of the tumor cell in vivo is different from cell line in vitro very much .Moreover, tumor tissues, especially HCC, are highly heterogeneous compared to cell lines. So it is urgent to establish proteomics technologies suitable for HCC tissue.In this paper, we constructed the basic method of HCC tissue treatment for 2D analysis and compared influence of 4 different sample treatments of freshly-prepared HCC tissues on 2D profile .We found that, currently, hepatic cell pool based on Perocoll density-gradient separation is relatively the best method of HCC tissue treatment for 2D analysis.更多还原