【作者】 伍龙；

【导师】 李雁；

【作者基本信息】 武汉大学 ， 肿瘤学， 2010， 博士

【摘要】 肝癌是最常见的恶性肿瘤之一,每年大约有60万人死于肝癌,居癌症死亡人数第三位。亚洲和撒哈拉以南非洲的肝癌发病率最高。由于HCV感染,估计未来10-20年西方的肝癌发病率增加2倍。对于早期和局部病灶而言,手术切除仍是可能获得治愈的主要手段。即使如此,根治术后5年的复发率仍达70%。尽管近年来在肝癌诊治和机理研究方面取得了长足的进步,临床可用的预测肝癌侵袭和转移的标志物仍然缺少。不同转移潜能的人肝癌细胞MHCC97L和HCCLM9细胞是研究肝癌的良好模型系统。HCCLM9为肝癌高转移细胞,由体内筛选而来,MHCC97L为肝癌低转移细胞。两者遗传背景相仿,转移潜能不同。细胞膜分隔胞内容物和外环境,在维持细胞功能方面扮演着重要的作用。膜蛋白和其他组分共同构成细胞结构,维持细胞间的运动和识别,参与结合受体,细胞内外的运输,细胞间和细胞基质间的相互作用,以及构成细胞骨架。当肿瘤生物学行为改变时,癌细胞膜蛋白的组分和特性也会发生改变,这有可能是之成为潜在的标志物。血清是获得肿瘤标志物的理想来源。肿瘤细胞与微环境相互作用后,从原发瘤转移至远处器官。来自病灶的蛋白裂解物进入淋巴和血液,随之可能在血清和组织中检出,成为潜在的肿瘤标志物。本研究首次采用蛋白质谱技术,分析不同转移潜能的人肝癌模型MHCC97L和HCCLM9的膜蛋白和血清,对差异蛋白进一步行动物模型及临床标本验证,旨在寻找与肝癌侵袭转移相关的靶分子。第一部分：不同转移潜能人肝癌细胞膜蛋白质组学研究目的采用膜蛋白质组学技术筛选与原发性肝癌侵袭和转移相关分子,并加以验证。方法以HCCLM9和MHCC97L不同转移潜能人肝癌细胞为研究对象,采用SDS-PAGE和ESI-MS/MS技术比较和鉴定差异表达的细胞膜蛋白,进一步行western blot、动物模型标本和临床标本验证。结果MHCC97L与HCCLM9相比,显著性差异表达细胞膜蛋白14个,其中coronin-1C在HCCLM9细胞表达升高2.57倍,在裸鼠肝癌组织中免疫组化提示coronin-1C表达也明显升高。115例临床病理组织免疫组化显示,coronin-1C表达越强,肿瘤的侵袭程度越强、临床病期越晚。结论coronin-1C与肝癌侵袭转移相关。第二部分：不同转移潜能人肝癌模型血清蛋白质组学研究目的采用血清蛋白质组学技术筛选并鉴定肝癌转移相关的蛋白分子。方法建立不同转移潜能人肝癌HCCLM9和MHCC97L动物模型各8只,采用双向凝胶电泳和MALDI串联飞行时间质谱仪(MALDI-TOF/TOF)技术比较和鉴定差异表达的血清蛋白分子,western blot和动物模型标本验证后,建立肝癌肺转移裸鼠模型,确认目的蛋白在肿瘤转移过程中的变化,进一步对208例临床标本(男性170例,女性38例)验证。结果HCCLM9和MHCC97L组相比,血清蛋白表达显著差异达11个,其中补体因子H(complement factor H, CFH)在HCCLM9模型血清中水平(213.83±55.17)比MHCC97L组(122.48±48.91)高1.75倍。血清western blot结果显示当肝癌肺转移发生时,CFH的表达达到顶峰。裸鼠肝癌组织中免疫组织化学提示CFH表达明显升高。208例临床病理组织显示,CFH表达越强,临床病期越晚。结论CFH与肝癌侵袭转移相关。更多还原

【Abstract】 Hepatocellular carcinoma (HCC), accounting for an estimated 600,000 deaths annually, is the third leading cause of cancer-related mortality worldwide. Most cases occur in Asia and sub-Saharan Africa, however, the incidence is also expected to double over the next 10 to 20 years in the West, possibly due to the increased HCV infection. While curative therapies are possible if the lesion remains early and localized, almost 70% of resected cases recurred within 5 years. Impressive progression has been made in providing an increasingly comprehensive portrayal of HCC. However, biomarkers that indicate the risk of invasion and metastatic potential of HCC and can be widely used in clinical settings are not currently available.For a better insight into the characteristic of HCC metastasis, the stepwise metastatic human HCC cells MHCC97L and HCCLM9, with low and high metastatic potentials, were established via repeated in vivo selection and characterized by a similar genetic background but with significant differences in spontaneous metastasis behavior, providing appropriate model systems for comparative study on the molecular events correlated with HCC metastasis.Plasma membrane, the structure surrounding all living cells and acting as the primary interface between the cellular contents and the extracellular environment, plays crucial roles in cell functions. Membrane proteins and other components maintain cell structure, motility and recognition involved in receptor-binding and further transport of bound components into the cell, cell-cell and cell-matrix interactions, and the organization of the cytoskeleton. The composition and characteristics of membrane proteins of tumor cells are modified during malignant transformation and make them likely candidates for cancer biomarkers.Serum and tissue are optimal sources for discovery and analysis of cancer biomarker. Metastasis is the result of cancer cell adaptation to a tissue microenvironment at a distance from the primary tumor. Proteins derived from diseased tissue compartments may leak into lymph and blood, eventually becoming detectable in serum and tissues and representing potential biomarkers of disease.The current work was firstly to identify potential proteins from cell membrane and serum related to HCC invasive progression, using human HCC cells with different metastasis potentials, by proteomics analysis, experimental animal studies and clinical validation.Part One:Proteome analysis of cell membrane from hepatocellular carcinoma model system with multiple metastatic potentialsObjective To search for hepatocellular carcinoma (HCC) invasion and metastasis related biomarkers using the cell membrane proteomics approaches, and to validate the markers using experimental and clinical specimens. Methods HCCLM9 and MHCC97L cell, with a similar genetic background and remarkably different metastasis behaviors, were used for comparative membrane proteome profiling using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrospray ionization mass spectrometry (ESI-MS) technologies. Candidate protein makers were further validated by Western blot on cells, immunohistochemistry (IHC) on animal tumor tissues, and tissue micro-array on clinical specimens. Results Membrane proteins from MHCC97L and HCCLM9 cells were compared by SDS-PAGE analyses. A total of 14 proteins were identified by ESI-MS/MS among the differential bands. Coronin-1C was overexpressed in HCCLM9 (7.31±0.73) versus MHCC97L (2.84±0.99), and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Elevated coronin-1C expression was observed in liver cancer tissues of HCCLM9 nude mice. IHC study in 115 human HCC specimens demonstrated that patients with higher coronin-1C expession showed greater invasiveness and more advanced stage. Conclusion The study suggests coronin-1C could be a candidate biomarker to predict HCC invasive behavior.Part One:Proteome analysis of serum from hepatocellular carcinoma model system with multiple metastatic potentialsObjective Serum proteomics approaches were applied to search for and validate hepatocellular carcinoma (HCC) invasion and metastasis related biomarkers. Methods 16 male athymic BALB/c nu/nu mice were randomly divided into two groups. HCCLM9-and MHCC97L-nude mice models of human HCC, with a similar genetic background and remarkably different pulmonary metastasis potential, were established.2D gel electrophoresis (2-DE) and MALDI-TOF/TOF technologies were used to construct a comparative proteome profile of nude mice sera from HCCLM9-and MHCC97L-nude mice. Candidate protein was confirmed by western blot and measured in sera from tumor development of spontaneous pulmonary metastasis in HCCLM9-nude mice. This result was further was analyzed by immunohistochemistry (IHC) on animal tumor tissues, and tissue micro-array on clinical specimens including 170 male cases and 38 female cases. Results Serum proteins from HCCLM9-and MHCC97L-nude mice were compared by 2-DE. A total of 11 proteins were identified by MALDI-TOF/TOF. Complement factor H (CFH) was overexpressed in HCCLM9-(213.83±55.17) versus MHCC97L-nude mice serum (122.48±48.91) by western blot, and validated by IHC from both nude mice tumor tissues and clinical specimens. Serum CFH level showed a remarkable upsurge when pulmonary metastasis occurred. Elevated CFH expression was observed in liver cancer tissues of HCCLM9 nude mice. IHC study in 208 human HCC specimens including demonstrated that patients with higher CFH expession showed greater invasiveness and more advanced stage. Conclusion The study suggests CFH could be a candidate biomarker to predict HCC invasive behavior.更多还原