【作者】 邹奇飞；

【导师】 沈锋；

【作者基本信息】 第二军医大学 ， 外科学， 2009， 博士

【摘要】 肝癌是我国常见的恶性肿瘤之一,我国肝癌死亡率高居世界之首。虽然近半个世纪来肝癌的临床诊断和治疗取得了长足进步,但其远期疗效仍不满意,主要原因是术后肿瘤的复发和转移。肝癌根治性切除后其5年复发率高达61.5%。为此,肝癌转移复发的研究已引起广泛重视,成为目前肝癌防治工作中的重点。肝癌的转移是一个多环节,多阶段的过程。其过程包括:瘤细胞从原发灶脱落,降解基底膜,通过迁移进入血管、淋巴管,进而进入循环,瘤细胞黏着于远处毛细血管内皮细胞和暴露的基底膜,在远隔器官内侵袭、增殖,最终形成远处转移灶。通过对肝癌患者原发灶和转移灶的基因芯片研究发现,两者之间有着相似的基因表达,这提示我们,这部分引起转移的肿瘤细胞具有复制出和原发灶异质性相同的能力。目前对恶性肿瘤的研究发现,肿瘤组织或者肿瘤转移灶中存在极少的且具有无限自我更新能力并可导致肿瘤发生的细胞-肿瘤干细胞(cancer stem cells,CSCs)。在对乳腺癌和胰腺癌的研究中发现,CSC是引起肿瘤转移的重要原因。肝癌中肿瘤干细胞的研究才刚刚起步,目前认为,肝癌干细胞的来源可能为肝干细胞或肝祖细胞,即卵圆细胞。最近,一些研究人员利用肿瘤干细胞表面标志物CD133,从肝癌组织及肝癌细胞系中分离出具有干细胞特性的亚群,为肝癌干细胞的存在找到了新的证据。这些肝癌组织中CD133+细胞,不仅在体外具有很强的增殖和克隆形成能力,还在免疫缺陷鼠的体内具有明显的成瘤优势;同时,CD133+细胞具有较高的干细胞相关基因如catenin、Oct-3/4、Bmi、SMO、Notch-1的表达;在体外适当条件下培养,CD133+细胞能分化为血管内皮样,骨骼肌样或心肌样细胞。那么,CD133+肿瘤干细胞是不是导致肝癌转移的主要原因?是不是所有的CD133+肿瘤干细胞都可以引起肝癌转移呢?本实验研究CD133+肿瘤干细胞在肝癌转移中的作用。通过对肝癌组织的免疫组织学检查发现,肝癌中CD133的表达和肿瘤转移相关。在体外,CD133+肝癌细胞具有较强的增殖能力,但并未表现出明显的侵袭方面的优势。另外,我们发现,粘附分子CD44在有转移的肝癌组织和肿瘤侵袭边缘表达较高,而CD44+细胞具有较强的体外侵袭能力但并具有较强的增殖优势。结合上述两个分子,我们发现,肝癌中存在着CD133+CD44+的肿瘤细胞,并且其比例和肿瘤转移相关。并且,在体内只有CD133+CD44+肝癌细胞能引起肿瘤的肝内和肝外肺转移。综上,我们认为,肝癌中CD133+细胞具有肿瘤干细胞的特性,而其中一部分特殊的肿瘤干细胞群体,表达为CD133+CD44+,是引起肝癌转移的重要原因。这部分特殊的肿瘤干细胞群体可以作为临床抗肝癌复发和转移的新的靶点。全文分为三个部分。第一部分,肝癌中,CD133+肿瘤干细胞具有较强增殖和成瘤能力,但是并未表现出明显的侵袭和转移方面的优势。首先,用免疫组化的方法检测肝癌组织中存在少量CD133+的细胞,然后用流式细胞方法从肝癌细胞系中分离出CD133+细胞,观察其体外克隆形成能力以及抗化疗药物能力,并通过RNAi干扰证实,CD133具有一定的功能,并非是一个单纯的表面标志物。但在体外侵袭迁移实验中,CD133+细胞并未表现出这方面的优势,因此推断,CD133在肿瘤转移成瘤中起着一定作用,但并不代表肿瘤细胞侵袭方面的能力,可能还需要另外一个信号通路来协助肿瘤干细胞的向外侵袭和转移。第二部分,CD44在肝癌细胞的侵袭中起着重要作用。同样,这部分首先用组化的方法检测肝癌组织中CD44的表达,发现其主要在肿瘤侵袭边缘表达,并且和癌栓及肝内转移相关。肝癌细胞系中,CD44+细胞具有明显的体外侵袭优势,但体外克隆形成能力和CD44-组比较无明显差别。提示CD44的表达利于肿瘤的侵袭。第三部分,CD133+CD44+肿瘤细胞是肝癌侵袭和转移的主要来源。无论是免疫组化还是流式细胞技术,都检测到肝癌组织中存在着CD133+CD44+的肿瘤细胞,并且和肝癌癌栓形成及肿瘤分期相关。从肝癌细胞系中分离出的CD133+CD44+细胞,在裸鼠体内实验中较其他组分表现出更强的肝内和肝外转移能力,从而证明这部分细胞是负责肝癌侵袭和转移的重要因素。全文结论:肝癌组织中,CD133可以作为肿瘤干细胞标志物,在肝癌的成瘤、增殖及抗凋亡方面起着重要作用;CD44则是肝癌侵袭能力的标志,其表达有助于肝癌细胞向外侵袭和转移。而肝癌的肝内及肝外转移则需要上述两个信号通路的同时激活,即CD44的表达利于肿瘤细胞的向外运动和侵袭,CD133的表达则利于肿瘤细胞在肝内外的增殖和生长。更多还原

【Abstract】 Liver cancer is one of the most common malignant tumor in our country ,and it’s mortality is the highst in the world.though the clinic diagnosis and treatment have made great improvement in last half century,the long term curative effect remain still unsatisfied .,the most important reason is tumor metastasis and relapse .the 5-year recurrent rate of liver cancer is as high as 61.5% after radical resection.so study on the recurrence and metastasis has brought about extensive attention in our country and become a emphasis in tumor prevention at present. The metastasis of liver cancer is a multi-step process including falling of tumor cells from the primary tumor, degradation of the base membrane,invasion into the circulation,anchoring on the target organ , proliferation and growth into metastasis tumor. Through gene microarray on primary tumor and accordance metastasis of liver cancer ,we find similar gene profile between the two samples. It’s suggest that the tumor cells formed metastasis have the ability to copy the heterogeneity of cells in primary tumor.In the study on malignant tumors ,a rare cell population defined as cancer stem cells(CSCs) in the primary and metastasis has been found which has the ability of unlimited selfrenew and tumorformation . moreover in the research on breast cancer and pancreatic cancer we found that the CSC could induce tumor metastasis. Research on CSC in liver cancer is just in a development stage. And the liver cancer stem cells are believed to origin from liver stem cells or liver progenitor cells at present . recently , by the cell surface marker CD133,some researchers separated a subpopulation possessed the character of cancer stem cells from liver cancer tissue and cell lines. It’s found a new proof of the presence of CSCs in liver cancer. These CD133+ tumor cells not only possess extensive capability of proliferation and clonformation in vitro,but appear advantages of tumorformation in immunodeficiency mice. And some stem cell-related genes such as catenin,oct-3/4.bmi,notch-1 could be found highly expressed in these cell proliferation .cultured in proper condition in vitro,these cells could differentiate into endothelia cells and skeletal muscle cells or myocardial cells.Whether these CD133+ tumor cells mediate liver cancer metastasis? And whether all the CD133+ cancer stem cells could induce metastasis? Our research lay emphasis on the role of CD133+ cancer stem cells in liver cancer metastasis. Through immuno-histological analysis of human HCC samples, we found the increased expression of CD133 correlated with HCC metastasis. Although CD133+ tumor cells displayed an increased proliferation rate, they were not advantageous in cell invasion in vitro. We then identified CD44+/high as a marker to isolate a high invasive population in HCC cells in vitro. CD44+ tumor cells were found more in metastatic HCC samples and enriched in the tumor boarder. We then combined CD133+ and CD44+/high to define a putative metastatic population in HCC cells. CD133+CD44+ tumor cells were found more abundant in metastatic HCC samples, compared with those in non-metastatic HCC samples. These cells were present in both primary tumor bulk and their derived tumor thrombus. Moreover only CD133+CD44high HCC cells could generate intrahepatic and lung metastasis in animal models. Down-regulating CD133 or CD44 led to inhibition of the liver cancer growth or invasion, respectively. Taken together, we here identified a subpopulation of liver cancer stem cells, characterized by the CD133+CD44+/high phenotype, which mediated the tumor metastasis and these cells might be a new target for treatment of HCC metastasis. This study is civided into 3 parts.Part one,the role CD133+ tumor cells in liver cancer. First ,by IHC we detected a rare population of CD133+ cells in liver cancer samples,and their proportions are corelatrd with tumor metastasis. then we separated CD133+ cells from HCC cell lines by flow cytometry. The CD133+ cancer cells appared stem cell characters in comparing their capability of cloneformation and drug-resistant. by RNAi,we found its functional role of CD133+ cells in HCC tumor formation. Whereas we do not find its invasion advantage through transwell in vitro. So we make a conclution that CD133+ tumor cells might be the cancer stem cells in HCC, but do not contribute to cancer cells invasion.Part two,CD44 play a important role in HCC metastasis. Also by IHC ,we found the expression of CD44 in HCC tissue mostly at the edge of invasion ,and had relations with tumor embolism and intrahepatic metastais. By clone formation and charmber invasion assay, we detected that CD44+ cancer cells had marked advantage of invasion but not proliferation. So we conclude that the expression of CD44 took advantage of tumor invasion and metasitasis.Part three ,CD133+CD44+ tumor cells mediate HCC invasion and metastasis. We could detect CD133+CD44+ tumor cells in primary tumor and metastasis with immunofluorescence or flow cytometry, and the percentage of these cells have relations with tumor embolism and tumor stage. Moreover only CD133+CD44+ tumor cells separated from HCC cell lines could mediate intrahepatic or lung metastasis in nude mice. Here we prove CD133+CD44+ cancer cells play an important role in HCC metastasis. Conclusion: in HCC, CD133 may be the cells surface marker of CSCs, and help tumor formation, proliferation and apoptosis-resistant. While CD44 can increase the invasive ability of tumor cells. It’s necessary for the cancer cells to combine these two molecules to mediate intrahepatic and lung metastasis in HCC.更多还原