肝癌细胞与成纤维细胞的相互作用对肝癌肺转移的影响

发布时间：2018-06-01 01:04

本文选题：肝癌 + 肿瘤相关成纤维细胞　；参考：《第二军医大学》2017年博士论文

【摘要】：研究背景和目的原发性肝癌是世界范围内第五大常见的恶性肿瘤,死亡率极高,严重威胁了人类的健康。我国的肝癌患者占世界的一半以上,且80%的患者处于中晚期,预后差。肝癌的主要致死原因是侵袭和转移,而在肝癌的远隔转移中,肺转移占主要部分。“种子-土壤”学说认为,肿瘤的转移过程与肿瘤微环境关系密切。因此,进一步深入研究肝癌微环境对肝癌肺转移的影响,对临床靶向肺转移的预防和治疗具有潜在的重要意义。肿瘤微环境是指肿瘤发生发展过程中所处的内环境,包括肿瘤细胞、成纤维细胞、免疫细胞、内皮细胞和多种细胞因子等成分。肿瘤细胞与微环境中非肿瘤细胞之间的交互作用,是肿瘤细胞调控肿瘤微环境的重要过程,对肿瘤细胞的生存和发展有着极其重要的意义。在非肿瘤细胞中,成纤维细胞是主要成分,其活化形式即肿瘤相关成纤维细胞,能够为肿瘤生长提供营养支持和多种活性因子,参与肿瘤的增殖、侵袭转移和耐药等多个方面的调控。在肿瘤微环境中,除了细胞因子发挥作用之外,外泌体作为重要的信息媒介,成为近年来研究的热点。外泌体是细胞生成的一种直径在30到100纳米的囊泡,其内包含有多种核酸、蛋白质和脂质等物质,是介导肿瘤微环境中多种细胞之间生物信息交流的重要介质,在肿瘤的转移、耐药等方面发挥着重要的调控作用。然而,在肝癌肺转移过程中,肝癌细胞如何调控肿瘤微环境以促进肺转移形成,目前尚不清楚。尤其是肝癌细胞与成纤维细胞之间交互作用对肺转移形成的影响,以及肝癌微环境中外泌体对介导肝癌肺转移过程的影响均无系统研究阐明。并且,肝癌细胞在肺转移形成能力上存在较大异质性,这一点是否与肝癌细胞对微环境特别是成纤维细胞的教化能力不同有关,亦缺少相关研究证实。因此,深入研究肝癌细胞对微环境中成纤维细胞的调控及其机制,对理解肝癌肺转移异质性,在临床治疗及预后判断方面具有重要价值。本研究基于肝癌肺转移存在异质性这一现象,系统研究了不同转移能力的肝癌细胞对成纤维细胞的教化能力差异,提出了高转移肝癌细胞与成纤维细胞交互作用的具体机制。本研究将有助于进一步理解肝癌肺转移的具体过程,为临床肝癌肺转移的预防和治疗提供新的思路。研究方法1、选取常见四种肝癌细胞并通过尾静脉注射建立体内肺转移模型,根据肺转移形成能力高低,分为高转移肝癌细胞和低转移肝癌细胞;2、在体外,收集肝癌细胞条件培养基及外泌体,分别刺激人胚肺成纤维细胞,比较转移能力差异对成纤维细胞招募及激活能力差异的影响,以及外泌体在肝癌细胞教化成纤维细胞过程的作用;3、通过尾静脉注射建立肝癌细胞SMMC-7721的肺转移模型,随机分组后分别给予所选四种肝癌细胞的外泌体及对照处理,比较不同转移能力肝癌细胞来源的外泌体对肝癌肺转移的影响;4、通过收集所选四种肝癌细胞的外泌体并进行miRNA芯片测试,分析其表达差异并结合功能实验筛选高转移肝癌细胞教化成纤维细胞过程中的关键miRNA;5、通过miRNA靶点预测并结合功能实验,筛选上述关键miRNA在成纤维细胞中发挥活化作用的直接靶点;6、通过蛋白免疫印迹技术及报告基因实验,进一步研究该miRNA在成纤维细胞中发挥作用的主要信号通路;7、通过建立肿瘤相关成纤维细胞体外诱导模型,研究其对肝癌细胞干性、EMT及肿瘤生长的影响;8、通过分离培养原代肿瘤相关成纤维细胞,研究其对肝癌细胞干性、EMT及肿瘤生长的影响;9、收集临床肝癌组织及血清样本,检测上述miRNA的临床样本中的表达情况及对临床预后的相关性,分析血清外泌体中该miRNA的表达与肝癌肺转移的相关性。结果1、高转移能力的肝癌细胞具有更强的招募及活化成纤维细胞以促进肝癌肺转移的能力;2、高转移肝癌细胞来源的外泌体具有更强的招募及活化成纤维细胞的能力,继而促进肺转移的形成;3、高转移肝癌细胞生成含有miR-1247-3p的外泌体介导了肺转移微环境中的正常成纤维细胞的活化;4、β1,4亚基半乳糖转移酶3是肿瘤细胞教化成纤维细胞过程中miR-1247-3p的直接作用靶点;5、miR-1247-3p通过靶向β1,4亚基半乳糖转移酶3,继而上调β1型整合素-NF-κB信号通路,以激活成纤维细胞;6、活化后的成纤维细胞中IL-6和IL-8分泌能力增强,促进肝癌细胞的干性、EMT和肿瘤生长;7、血清外泌体中mi R-1247-3p的表达与肝癌肺转移呈正相关关系。结论本研究通过不同转移能力肝癌细胞对成纤维细胞的教化实验,发现高转移肝癌细胞具有更强的招募并激活成纤维细胞的能力。具体机制为,高转移肝癌细胞能特异性生成含有大量mi R-1247-3p的外泌体,后者被成纤维细胞摄取后,miR-1247-3p通过靶向β1,4亚基半乳糖转移酶3,进而上调β1型整合素-NF-κB信号通路,从而激活成纤维细胞。活化后的肿瘤相关成纤维细胞中IL-6和IL-8分泌能力增强,促进肝癌细胞的干性、EMT及肿瘤生长。在临床肝癌患者的血清外泌体样本中,miR-1247-3p的表达与肝癌肺转移具有正相关关系。本研究证实了,在肿瘤微环境中,上述高转移肝癌细胞与成纤维细胞的交互作用促进了肝癌肺转移进展,有助于为临床肝癌肺转移的预防和治疗提供新的方向。
[Abstract]:Background and objective primary hepatocellular carcinoma (HCC) is the fifth most common malignant tumor worldwide, with high mortality and serious threat to human health. In China, liver cancer accounts for more than half of the world, and 80% of the patients are in the middle and late stages, and the prognosis is poor. The main cause of the death of HCC is invasion and metastasis, and in the distant metastasis of liver cancer, the lung is in the lung. The "seed soil" theory holds that the metastasis process of the tumor is closely related to the tumor microenvironment. Therefore, it is of great significance to further study the effect of liver cancer microenvironment on the lung metastasis of liver cancer, which is of potential significance for the prevention and treatment of clinical target lung metastasis. The internal environment, including tumor cells, fibroblasts, immune cells, endothelial cells and a variety of cytokines, and the interaction between tumor cells and non tumor cells in the microenvironment, is an important process for tumor cells to regulate the tumor microenvironment and is of great significance to the survival and development of tumor cells. In the cell, fibroblasts are the main components, and their activation forms, tumor related fibroblasts, can provide nutritional support and a variety of active factors for tumor growth, participate in the regulation of tumor proliferation, invasion and metastasis and drug resistance. In the tumor microenvironment, the exocrine is an important letter in addition to the role of cytokines. Interest media has become a hot spot in recent years. Exosome is a cell - generated vesicle with a diameter of 30 to 100 nanometers. It contains a variety of nucleic acids, proteins and lipids. It is an important medium for the exchange of biological information among various cells in the tumor microenvironment, and plays an important role in the metastasis and resistance of the tumor. However, it is not clear how hepatoma cells regulate the tumor microenvironment to promote the formation of lung metastasis in the process of lung metastasis of liver cancer, especially the influence of the interaction of hepatoma cells and fibroblasts on the formation of lung metastasis, and the effect of the secreting body on the lung metastasis of liver cancer in the microenvironment of liver cancer. The study clarifies that there is a large heterogeneity in the ability of liver cancer cells in the formation of lung metastasis, whether it is related to the different teaching ability of hepatoma cells to microenvironment, especially fibroblasts, and is lack of relevant research. Therefore, the study of the regulation and mechanism of hepatoma cells in the microenvironment of fibroblasts and the understanding of liver The heterogeneity of cancer and lung metastasis is of great value in clinical treatment and prognosis. Based on the heterogeneity of the lung metastasis of liver cancer, this study systematically studies the differences in the teaching ability of hepatoma cells with different metastatic capacity to fibroblasts, and puts forward a specific mechanism for the interaction of high metastatic liver cancer cells and fibroblasts. This study will help to further understand the specific process of lung metastasis of liver cancer and provide new ideas for the prevention and treatment of lung metastasis in clinical liver cancer. Method 1, four kinds of common hepatoma cells were selected and the model of lung metastasis was established through the injection of the tail vein. The high metastatic liver cancer cells and low metastasis were divided into high metastatic liver cancer cells and low metastasis according to the lung metastasis. Hepatoma cells; 2, in vitro, the condition culture medium and external secretory of hepatoma cells were collected to stimulate human embryonic lung fibroblasts, and the difference in the difference of transfer ability on the recruitment and activation of fibroblasts, and the role of the exocrine in the process of fibroblasts in the liver cancer cells; and 3, the liver cancer cell SMMC was established by the injection of the tail vein. The lung metastasis model of -7721 was randomly assigned to the external secreting and control treatment of the selected four kinds of hepatoma cells, and the effect of external secreting on the lung metastasis of liver cancer was compared. 4. By collecting the exocrine of four kinds of hepatoma cells and testing the miRNA chip, the difference of expression and function were analyzed. To screen the key miRNA in the process of transforming high metastatic hepatoma cells into fibroblasts; 5, through the miRNA target prediction and functional experiment, the direct target of the key miRNA in fibroblasts was screened. 6, the miRNA in fibroblasts was further studied through protein immunoblotting and the report gene experiment. The main signaling pathways that play an important role; 7, by establishing an in vitro induced model of tumor related fibroblasts, the effects on the stem, EMT and tumor growth of hepatoma cells were studied. 8, the effects of the primary tumor related fibroblasts on the stem, EMT and tumor growth of the hepatoma cells were studied, and 9, to collect the clinical liver cancer tissue and Serum samples were used to detect the expression of miRNA in clinical samples and the correlation of clinical prognosis. The correlation between the expression of miRNA in serum exocrine and lung metastasis of liver cancer was analyzed. Results 1, high metastatic liver cancer cells have stronger recruitment and activation of fibroblasts to promote lung metastasis of liver cancer; 2, high metastasis liver The exosomes derived from cancer cells have a stronger ability to recruit and activate fibroblasts, and then promote the formation of lung metastases; 3, high metastatic liver cancer cells produce miR-1247-3p exosomes that mediate the activation of normal fibroblasts in the microenvironment of lung metastasis; 4, beta 1,4 subunit galactose transferase 3 is a neoplastic fibroblast The direct target target of miR-1247-3p during cell process; 5, miR-1247-3p through target beta 1,4 subunit half lactase 3, then up regulation of beta 1 integrin -NF- kappa B signaling pathway to activate fibroblasts; 6, enhanced secretion of IL-6 and IL-8 in the activated fibroblasts, promoting the stem, EMT and tumor growth of the hepatoma cells; 7, serum exocrine secretion. There is a positive correlation between the expression of MI R-1247-3p in the body and the lung metastasis of liver cancer. Conclusion this study shows that high metastatic liver cancer cells have a stronger ability to recruit and activate fibroblasts through the teaching experiments of different metastatic liver cancer cells to fibroblasts. The specific mechanism is that high metastatic liver cancer cells can produce a large number of M specifically. The exocrine of I R-1247-3p, which is taken by fibroblasts, is activated by miR-1247-3p by targeting beta 1,4 subunit galactose transferase 3, and then up regulation of the beta 1 integrin -NF- kappa B signaling pathway, thus activating fibroblasts. The secretion of IL-6 and IL-8 in the activated tumor related fibroblasts is enhanced to promote the dry, EMT, and tumor of the liver cancer cells. Growth. In the serum exocrine samples of patients with clinical liver cancer, the expression of miR-1247-3p has a positive correlation with the lung metastasis of liver cancer. This study confirms that the interaction of these high metastatic hepatoma cells and fibroblasts in the tumor microenvironment promotes the progression of lung metastasis of liver cancer and helps to prevent and treat the pulmonary metastasis of the liver cancer. Provide a new direction.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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