【作者】 傅修涛；

【导师】 樊嘉；

【作者基本信息】 复旦大学 ， 外科学， 2012， 博士

【摘要】 肝细胞肝癌(Hepatocellular carcinoma, HCC)(简称肝癌)是全球发病率和死亡率均较高的恶性肿瘤之一,其发病率居第五位,病死率居第三位,在我国每年新诊断的肝癌约占全世界的55%,其死亡率在我国恶性肿瘤中位列第二位。虽然近年来诊断和治疗技术的进步使得肝癌患者的生存率有了较大提高,但术后复发与转移已成为影响肝癌预后的主要因素。临床上需要探索和阐明肝癌复发转移的分子机制从而准确预测患者预后、寻找高效的靶点并设计合理干预治疗的新方法。传统的肝癌研究主要注重于肝癌细胞自身,探索其内在的基因和蛋白变化来解释肿瘤的复发和转移。然而,肿瘤是在一定的局部环境中生长的,所以局部的微环境对于肿瘤的发生发展也起着至关重要的作用。炎症微环境作为肿瘤微环境的重要组成部分,与肿瘤的发生发展、复发转移都有着密切的关系。例如乙肝病毒或丙肝病毒感染或诸如嗜酒、肥胖等非感染因素造成的肝脏慢性炎症及继发的肝硬化状态就是肝癌发生发展的“土壤”上皮-间质样表型转化(epithelial mesenchymal transition, EMT)是指在特定的生理和病理情况下,上皮细胞暂时丧失极性并表现出具有移行能力的间质细胞的现象,是癌细胞脱离原发灶完成转移的重要步骤。目前认为肿瘤细胞发生EMT是微环境中作用因子和肿瘤细胞相互作用的结果,这导致细胞通路和基因表达发生改变,最终使肿瘤细胞更富于侵袭性而利于转移。肿瘤相关巨噬细胞(Tumor associated macrophage, TAM)存在于肿瘤间质中,是肿瘤免疫微环境中主要的成分,其通过分泌特殊的细胞因子、生长因子等作用于肿瘤相关基质或内皮细胞,参与了促肿瘤发展、刺激肿瘤细胞生长和转移,诱导肿瘤新生血管和淋巴管生成、免疫抑制等过程。在我所前期研究中发现,肝癌患者癌旁组织中的巨噬细胞集落刺激因子(M-CSF)在肿瘤边缘浓度最高,癌旁组织中M-CSF和巨噬细胞增多与患者预后不良相关；在巨噬细胞与黑色素瘤的相关研究中,发现肿瘤侵袭边缘的CD68+巨噬细胞密度与患者预后负相关。这些现象提示可能在肿瘤侵袭边缘区域,巨噬细胞通过分泌某些细胞因子增加这些区域中肿瘤细胞的侵袭转移能力,而促进其复发及转移。目前,关于肿瘤相关巨噬细胞与人肝癌细胞EMT二者之间的联系尚无相关研究。本研究通过体外建立人巨噬细胞与人肝癌细胞系共培养系统,系统性地研究巨噬细胞对肝癌细胞发生EMT的影响及其相关分子机制。并通过抗体芯片的方法筛选可能起相关作用的细胞因子,进一步通过组织微阵列技术,对肝癌微环境中相关炎症因子的分析,确定主要的细胞因子,对可能的细胞信号通路进行验证,初步阐明在人肝癌组织中巨噬细胞引发肝癌细胞EMT的现象与机制,为肝癌的发生发展和治疗提供新的理论支持。本课题分为三部分：1、人巨噬细胞对不同转移潜能人肝癌细胞系生物学特性影响的研究；2、巨噬细胞对人肝癌细胞上皮-间质样表型影响的研究；3、巨噬细胞分泌的IL-8对人肝癌细胞上皮-间质样表型影响及相关机制的研究。第一部分人巨噬细胞对不同转移潜能人肝癌细胞系生物学特性影响的研究目的：研究人巨噬细胞对不同转移潜能人肝癌细胞系生物学特性的影响,初步探索巨噬细胞与人肝癌细胞系生物学特性改变的相关性。方法：首先将人白血病单核细胞系THP-1,经过佛波酯刺激后分化成为巨噬细胞；其次选择人高转移潜能肝癌细胞系MHCC-97H,MHCC-97L与人低转移潜能肝癌细胞系Hep-G2, Huh-7,于六孔板小室中与巨噬细胞共培养,观察其生物学形态改变；应用细胞划痕实验检测共培养体系中肝癌细胞运动迁移能力；应用Transwell侵袭小室检测共培养体系中肝癌细胞对的侵袭能力。结果：巨噬细胞与人肝癌细胞系共培养之后,细胞形态发生显著改变,运动迁移能力增强,对基底层的侵袭能力增强。结论：巨噬细胞可能通过某些细胞因子增强人肝癌细胞系的运动及迁移侵袭能力。第二部分巨噬细胞对人肝癌细胞上皮-间质样表型影响的研究目的：研究巨噬细胞对人肝癌细胞发生上皮-间质样表型转变的影响,探索二者之间的相关性及可能发挥作用的细胞因子。方法：随机选取30例普通肝癌患者手术病理石蜡切片,利用免疫双染的方法观察巨噬细胞与肝癌组织中发生上皮-间质样表型转变的细胞之间的关系；将高转移潜能人肝癌细胞系MHCC-97H、MHCC-97L与低转移潜能人肝癌细胞系Hep-G2、Huh-7与THP-1诱导分化的巨噬细胞共培养；应用WesternBlot的方法检测上皮-间质样变化的分子标志E-Cadherin、N-Cadherin和Snail(?)勺表达情况；应用免疫荧光的方法检测E-Cadherin、N-Cadherin和Snail表达的变化；应用抗体芯片的方法检测并筛选可能发挥作用的细胞因子。结果：在人肝癌组织切片中,巨噬细胞浸润区域中肝癌细胞间质样标志物N-Cadherin及Snail的表达增强；WesternBlot显示E-Cadherin的表达随着肝癌细胞系转移潜能的增高而减弱,N-Cadherin及Snail的表达随着肝癌细胞系转移潜能的增加而增强；与巨噬细胞共培养后,和对照组相比较,E-Cadherin表达减弱,而N-Cadherin与Snail表达增强；免疫荧光显示共培养组E-Cadherin表达低于对照组,N-Cadherin与Snail表达高于对照组：抗体芯片提示IL-6与IL-8可能为主要作用的细胞因子。结论：巨噬细胞可引起人肝癌细胞发生上皮-间质样表型的转变,该作用可能与IL-6和IL-8有关。第三部分巨噬细胞分泌的IL-8对人肝癌细胞上皮-间质样表型影响及相关机制的研究目的：通过研究人肝癌组织内IL-8与CD68的表达情况,并分析共培养系统中巨噬细胞分泌的IL-8的作用,探索其促进人肝癌细胞发生上皮-间质样表型转变的机制。方法：应用ELISA法分别检测人肝癌细胞系Hep-G2、人巨噬细胞及二者共培养体系中培养液上清中IL-8的浓度；应用组织芯片和免疫组化的技术比较IL-8、IL-6及巨噬细胞表面标志物CD68的表达,分析二者的相关性；应用免疫荧光的方法比较外源性IL-8作用于人肝癌细胞系Hep-G2后EMT标志物E-Cadherin、 N-Cadherin及Snail的变化情况；应用细胞划痕实验检测外源性IL-8对肝癌细胞运动迁移能力的影响；应用Transwell侵袭小室检测外源性IL-8对肝癌细胞侵袭能力的影响；应用WesternBlot的方法检测外源性IL-8对肝癌细胞上皮-间质样表型转化中信号通路的变化情况；结果：人巨噬细胞及其与人肝癌细胞系Hep-G2共培养体系中培养液上清中IL-8浓度显著高于人肝癌细胞系Hep-G2(P<0.001);IL-6在人肝癌癌内与癌旁组织中均有表达,其表达无个体与组织特异性,而IL-8与CD68二者在人肝癌癌内与癌旁组织中表达存在正相关性(P<0.0001)：外源性IL-8可促进肝癌细胞的运动迁移能力及侵袭能力；外源性IL-8可以通过JAK2/STAT3途径激活Snail.从而使细胞发生上皮-间质样表型转变。结论：巨噬细胞通过分泌IL-8作用于肝癌细胞使之发生上皮-间质样表型转变。创新点1.初步探索人巨噬细胞对人肝癌细胞EMT的影响及可能的作用机制。2.首次利用抗体芯片在人巨噬细胞与人肝癌细胞共培养体系中大量筛检相关细胞因子,初步阐明了IL-8在肝癌细胞EMT中的作用机制。潜在应用价值1.肝癌组织中巨噬细胞是一个潜在的肝癌转移复发的治疗靶点。2.为深入研究肝癌发生和转移过程中巨噬细胞参与肝癌侵袭转移的机制提供实验基础。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most prevalent human malignant tumors worldwide and especially in Asia and South Africa. Surgical intervention is the most common form of therapy of it, but the prognosis of HCC remains poor because of tumor invasiveness, frequent intrahepatic spread, and extrahepatic metastasis.The vast majority of previous studies focused solely on malignant cells themselves, regarding tumors just as masses of autonomous cells and aiming at identifying the molecular and genetic changes associated with this malignant transformation. Recent research on tumor-host interactions collectively reveals that microenvironments co-evolve during tumor initiation and progression. As an important part of tumor microenvironment, immune microenvironment plays a critical role in carcinogenesis, metastatic invasion and dissemination. HCC is usually secondary to inflammation conditions due to chronic hepatitis and cirrhosis resulting from either HBV/HCV infection or non viral-related causes such as alcohol or obesity.Metastasis is a complex process and various factors are involved in each step of metastasis. Recent studies suggest that numerous genes and proteins play vital roles in metastasis of cancer cells, and epithelial-mesenchymal transition (EMT) which describes that epithelial cells can convert into mesenchymal cells has been shown to be critical for the development of the invasiveness and metastatic potential of human cancers. During the transition process, epithelial cells reduce intercellular adhesion and acquire fibroblastoid propertis including down-regulation of E-Cadherin which is epithelial marker, up-regulation of N-Cadherin which is mesenchymal marker, and Snail which is the EMT regulator. EMT can help the cancer cells enter the surrounding tissues by increased invasive properties and finally establish a suitable microenvironment for its progression and metastasis. Tumor associated macrophage (TAM) is a special subset of tumor immune cells which is located in the tumor tissues and plays a critical role in tumor immune microenvironment. TAM can secret some cytokines and growth factors which involved in promoting tumor development, stimulate the growth and metastasis of tumor cells, induced tumor angiogenesis and lymphangiogenesis, immune suppression process. In our previous study we found that high peritumoral macrophage colony-stimulating factor (M-CSF) and macrophage density, especially at the tumor edge, predicted high risk of recurrence and metastasis in HCC patients who had received curative live resections. It is reported that dense CD68+macrophage infiltration at the tumor border line related to poor survival in melanoma, and in some other reports (bladder, esophageal, and breast cancer), the same phenomenon was observed. These phenomena suggest that in the area of the tumor edge, macrophages can increase the ability of invasion and metastasis of tumor cells through the secretion of certain cytokines, and promote their recurrence and metastasis.In this study, by establishing a macrophage-HCC cell lines co-culture system, we can perform a similar microenvironment of tumor edge in vitro. This model can assist us in exploring whether the macrophage promote the procedure of EMT. In order to observe the phenomenon in vivo, double staining immunohistochemistry and tissue micro-array was used to localize the biomarker of macrophage and EMT in specimens obtained from HCC patients simultaneously. Cell signal microarray was applied to discover the cytokines during the interaction between paritumoral macrophage and EMT.These experiments are divided into3parts, as following,1. Research on the biological characteristics of macrophages effected human HCC cell lines with different metastatic potential;2. Study on the role of macrophages in the epithelial-mesenchymal transition of HCC cells.3. Study on the effect and mechanism of macrophages secreted IL-8in the epithelial-mesenchymal transition of HCC cells. Part Ⅰ. Research on the Biological Characteristics of Macrophages Effected Human HCC Cell Lines with Different Metastatic PotentialObjective:To study the biological characteristics of macrophages effected human HCC cell lines with different metastatic potential, and explore the correlation of macrophages and the biological changes of characteristics in human hepatoma cell line.Method:Raise the THP-1human leukemia monocyte cell line, and make it differentiate into macrophages through PMA stimulation; Raise the high metastatic potential hepatocellular carcinoma cell lines:MHCC-97H, MHCC-97L and the low metastatic potential of hepatocellular carcinoma cell lines:Hep-G2, Huh-7, in six-well plates and co-cultured with macrophages throught the transwell system. A wound-healing assay was used to evaluated the ability of cell migration. Cell invasion assays were performed using24-well transwells precoated with Matrigel.Results:The human HCC cell lines which were co-cultured with macrophages were significantly changed in morphology and express enhanced wound closured rates and the ability of invasiveness.Conclusion:Macrophages may enhance the migration of invasive ability of the human HCC cell line by certain cytokines.Part Ⅱ. Study on the role of macrophages in the epithelial-mesenchymal transition of HCC cellsObjective:To study the role of macrophages in the epithelial-mesenchymal transition of human HCC cells and explore the potential relationship between them and the cytokines which probably have some effect on the certain process.Method:Randomly choose30HCC samples from the patients who have received curative resection in2009, and double staining immunohistochemistry was applied to observe the relationship between macrophages and the HCC cell which undergoing EMT in the tissues. The levels of E-Cadherin, N-Cadherin and Snail in HCC cell lines co-cultured with macrophages were detected by WesternBlot and immunofluorescence. Antibody micro-array was used to explore the cytokines which probably take part in the certain course.Result:The double staining immunohistochemistry showed that in the macrophages infiltrated area, the expression of N-Cadherin and Snail were increased. WesternBlot and immunofluorescence showed that HCC cell lines with high metastatic potential have low levels of E-Cadherin and high levels of N-Cadherin and Snail. By contrast, HCC cell lines with low metastatic potential have high levels of E-Cadherin and low levels of N-Cadherin and Snail. After co-cultured with macrophages, the HCC cell lines can obtain enhanced expression of N-Cadherin and Snail. The result of antibody micro-array revealed that IL-6and IL-8may be the major cytokines in the certain process.Conclusion:Macrophages can involve in the epithelial-mesenchymal transition of HCC cells through IL-6and IL-8.Part Ⅲ. Study on the effect and mechanism of macrophages secreted IL-8in the epithelial-mesenchymal transition of HCC cellsObjective:To investigate the mechanism of epithelial-mesenchymal transition in HCC cells, the expression the IL-8and CD68in human HCC tissues and the effect of macrophages secreted IL-8in the co-culture system was explored.Method:Tissue micro-array and immunohistochemistry were used to analyze the correlation among IL-6, IL-8and CD68. The level of IL-8in Hep-G2, macrophage and co-cultured system culture supernatants were measured by ELISA. Immunofluorescence was applied to explore the different expression of E-Cadherin, N-Cadherin and Snail in Hep-G2between the control group and exogenous IL-8group. A wound-healing assay was used to evaluate the ability of cell migration and cell invasion assays were performed using24-well transwells precoated with Matrigel in Hep-G2treated with exogenous IL-8. WesternBlot was applied to explore the cell signal pathway caused by exogenous IL-8.Result:IL-6in human HCC tissues had no specific expression, while IL-8and CD68were significant correlated. The levels of IL-8in culture supernatant in macrophage and the co-cultured system were extremely higher than that in Hep-G2(P<0.001). The exogenous IL-8can enhance the wound closure rates of Hep-G2and the promote the invasion of it. The process of epithial-mesenchymal transition can be started by the JAK2/STAT3pathway by activation of Snail.Conclusion:Macrophage secreted IL-8can start the process of EMT in HCC cells.更多还原