本文選題：肝癌 + 腫瘤相關成纖維細胞　； 參考：《第二軍醫(yī)大學》2017年博士論文

【摘要】：研究背景和目的原發(fā)性肝癌是世界范圍內(nèi)第五大常見的惡性腫瘤,死亡率極高,嚴重威脅了人類的健康。我國的肝癌患者占世界的一半以上,且80%的患者處于中晚期,預后差。肝癌的主要致死原因是侵襲和轉(zhuǎn)移,而在肝癌的遠隔轉(zhuǎn)移中,肺轉(zhuǎn)移占主要部分�！胺N子-土壤”學說認為,腫瘤的轉(zhuǎn)移過程與腫瘤微環(huán)境關系密切。因此,進一步深入研究肝癌微環(huán)境對肝癌肺轉(zhuǎn)移的影響,對臨床靶向肺轉(zhuǎn)移的預防和治療具有潛在的重要意義。腫瘤微環(huán)境是指腫瘤發(fā)生發(fā)展過程中所處的內(nèi)環(huán)境,包括腫瘤細胞、成纖維細胞、免疫細胞、內(nèi)皮細胞和多種細胞因子等成分。腫瘤細胞與微環(huán)境中非腫瘤細胞之間的交互作用,是腫瘤細胞調(diào)控腫瘤微環(huán)境的重要過程,對腫瘤細胞的生存和發(fā)展有著極其重要的意義。在非腫瘤細胞中,成纖維細胞是主要成分,其活化形式即腫瘤相關成纖維細胞,能夠為腫瘤生長提供營養(yǎng)支持和多種活性因子,參與腫瘤的增殖、侵襲轉(zhuǎn)移和耐藥等多個方面的調(diào)控。在腫瘤微環(huán)境中,除了細胞因子發(fā)揮作用之外,外泌體作為重要的信息媒介,成為近年來研究的熱點。外泌體是細胞生成的一種直徑在30到100納米的囊泡,其內(nèi)包含有多種核酸、蛋白質(zhì)和脂質(zhì)等物質(zhì),是介導腫瘤微環(huán)境中多種細胞之間生物信息交流的重要介質(zhì),在腫瘤的轉(zhuǎn)移、耐藥等方面發(fā)揮著重要的調(diào)控作用。然而,在肝癌肺轉(zhuǎn)移過程中,肝癌細胞如何調(diào)控腫瘤微環(huán)境以促進肺轉(zhuǎn)移形成,目前尚不清楚。尤其是肝癌細胞與成纖維細胞之間交互作用對肺轉(zhuǎn)移形成的影響,以及肝癌微環(huán)境中外泌體對介導肝癌肺轉(zhuǎn)移過程的影響均無系統(tǒng)研究闡明。并且,肝癌細胞在肺轉(zhuǎn)移形成能力上存在較大異質(zhì)性,這一點是否與肝癌細胞對微環(huán)境特別是成纖維細胞的教化能力不同有關,亦缺少相關研究證實。因此,深入研究肝癌細胞對微環(huán)境中成纖維細胞的調(diào)控及其機制,對理解肝癌肺轉(zhuǎn)移異質(zhì)性,在臨床治療及預后判斷方面具有重要價值。本研究基于肝癌肺轉(zhuǎn)移存在異質(zhì)性這一現(xiàn)象,系統(tǒng)研究了不同轉(zhuǎn)移能力的肝癌細胞對成纖維細胞的教化能力差異,提出了高轉(zhuǎn)移肝癌細胞與成纖維細胞交互作用的具體機制。本研究將有助于進一步理解肝癌肺轉(zhuǎn)移的具體過程,為臨床肝癌肺轉(zhuǎn)移的預防和治療提供新的思路。研究方法1、選取常見四種肝癌細胞并通過尾靜脈注射建立體內(nèi)肺轉(zhuǎn)移模型,根據(jù)肺轉(zhuǎn)移形成能力高低,分為高轉(zhuǎn)移肝癌細胞和低轉(zhuǎn)移肝癌細胞;2、在體外,收集肝癌細胞條件培養(yǎng)基及外泌體,分別刺激人胚肺成纖維細胞,比較轉(zhuǎn)移能力差異對成纖維細胞招募及激活能力差異的影響,以及外泌體在肝癌細胞教化成纖維細胞過程的作用;3、通過尾靜脈注射建立肝癌細胞SMMC-7721的肺轉(zhuǎn)移模型,隨機分組后分別給予所選四種肝癌細胞的外泌體及對照處理,比較不同轉(zhuǎn)移能力肝癌細胞來源的外泌體對肝癌肺轉(zhuǎn)移的影響;4、通過收集所選四種肝癌細胞的外泌體并進行miRNA芯片測試,分析其表達差異并結(jié)合功能實驗篩選高轉(zhuǎn)移肝癌細胞教化成纖維細胞過程中的關鍵miRNA;5、通過miRNA靶點預測并結(jié)合功能實驗,篩選上述關鍵miRNA在成纖維細胞中發(fā)揮活化作用的直接靶點;6、通過蛋白免疫印跡技術(shù)及報告基因?qū)嶒?進一步研究該miRNA在成纖維細胞中發(fā)揮作用的主要信號通路;7、通過建立腫瘤相關成纖維細胞體外誘導模型,研究其對肝癌細胞干性、EMT及腫瘤生長的影響;8、通過分離培養(yǎng)原代腫瘤相關成纖維細胞,研究其對肝癌細胞干性、EMT及腫瘤生長的影響;9、收集臨床肝癌組織及血清樣本,檢測上述miRNA的臨床樣本中的表達情況及對臨床預后的相關性,分析血清外泌體中該miRNA的表達與肝癌肺轉(zhuǎn)移的相關性。結(jié)果1、高轉(zhuǎn)移能力的肝癌細胞具有更強的招募及活化成纖維細胞以促進肝癌肺轉(zhuǎn)移的能力;2、高轉(zhuǎn)移肝癌細胞來源的外泌體具有更強的招募及活化成纖維細胞的能力,繼而促進肺轉(zhuǎn)移的形成;3、高轉(zhuǎn)移肝癌細胞生成含有miR-1247-3p的外泌體介導了肺轉(zhuǎn)移微環(huán)境中的正常成纖維細胞的活化;4、β1,4亞基半乳糖轉(zhuǎn)移酶3是腫瘤細胞教化成纖維細胞過程中miR-1247-3p的直接作用靶點;5、miR-1247-3p通過靶向β1,4亞基半乳糖轉(zhuǎn)移酶3,繼而上調(diào)β1型整合素-NF-κB信號通路,以激活成纖維細胞;6、活化后的成纖維細胞中IL-6和IL-8分泌能力增強,促進肝癌細胞的干性、EMT和腫瘤生長;7、血清外泌體中mi R-1247-3p的表達與肝癌肺轉(zhuǎn)移呈正相關關系。結(jié)論本研究通過不同轉(zhuǎn)移能力肝癌細胞對成纖維細胞的教化實驗,發(fā)現(xiàn)高轉(zhuǎn)移肝癌細胞具有更強的招募并激活成纖維細胞的能力。具體機制為,高轉(zhuǎn)移肝癌細胞能特異性生成含有大量mi R-1247-3p的外泌體,后者被成纖維細胞攝取后,miR-1247-3p通過靶向β1,4亞基半乳糖轉(zhuǎn)移酶3,進而上調(diào)β1型整合素-NF-κB信號通路,從而激活成纖維細胞�；罨蟮哪[瘤相關成纖維細胞中IL-6和IL-8分泌能力增強,促進肝癌細胞的干性、EMT及腫瘤生長。在臨床肝癌患者的血清外泌體樣本中,miR-1247-3p的表達與肝癌肺轉(zhuǎn)移具有正相關關系。本研究證實了,在腫瘤微環(huán)境中,上述高轉(zhuǎn)移肝癌細胞與成纖維細胞的交互作用促進了肝癌肺轉(zhuǎn)移進展,有助于為臨床肝癌肺轉(zhuǎn)移的預防和治療提供新的方向。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.7

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