外泌体在肝细胞癌索拉菲尼抵抗及转移中的作用和机制研究

发布时间：2018-03-17 22:22

本文选题：肝细胞肝癌　切入点：外泌体　出处：《南京大学》2017年博士论文　论文类型：学位论文

【摘要】：肝细胞癌(Hepatocellular carcinoma,HCC)是临床常见的恶性肿瘤之一,位列全球范围肿瘤相关性死因第二位。HCC患者预后较差原因主要在于患者临床诊断时病期已较晚,肿瘤出现肝内或远处器官转移。索拉非尼是目前唯一批准用于HCC治疗的多靶点药物,HCC患者出现索拉菲尼抵抗也是影响其生存期的主要问题。既往研究发现,外泌体作为多种不同类型细胞分泌的纳米囊泡,可通过转运多种生物活性分子至靶细胞,参与肿瘤微环境的调控,进而影响肿瘤进展、转移及药物抵抗等。此外,肿瘤细胞来源的外泌体携带其来源细胞特异性的蛋白、脂类和miRNA等,因而将外泌体作为多种肿瘤诊断的生物标志物越来越受到人们重视。然而,目前关于不同侵袭特性HCC细胞来源的外泌体在HCC索拉菲尼抵抗以及EMT引起的肿瘤转移方面的作用尚未见报道。此外,外泌体关键miRNA在HCC诊断以及肿瘤发展中的具体作用和机制报道较少。本研究以HCC细胞来源的外泌体为研究重点,探讨不同侵袭特性HCC细胞来源的外泌体在HCC索拉菲尼抵抗、侵袭转移和临床诊断中的潜在作用和具体分子机制。我们首先通过超速离心方法提取了不同侵袭特性HCC细胞培养上清内的外泌体,并应用TEM、western blot等方法进行分析、鉴定。然后将受体HCC细胞与索拉菲尼及不同侵袭特性HCC细胞来源的外泌体共培养,探讨HCC细胞来源的外泌体与肿瘤细胞索拉菲尼抵抗的关系。我们发现不同侵袭特性HCC细胞来源的外泌体可在体内和体外诱导HCC细胞索拉菲尼抵抗,且高侵袭HCC细胞来源的外泌体效果更强。HCC细胞来源的外泌体可能通过逆转索拉菲尼引起的细胞凋亡以及激活HGF/c-Met/Akt通路,上调多种药物抵抗相关蛋白发挥此作用。然后,我们研究了不同侵袭特性HCC细胞来源的外泌体在肿瘤细胞侵袭、转移中的作用和机制。我们通过细胞划痕、Transwell等实验研究不同侵袭特性HCC细胞来源的外泌体对受体细胞迁移、侵袭能力以及细胞表型蛋白的影响。并进一步通过应用外源性TGF-β及其抑制剂LY2109761处理细胞后提取的外泌体验证以上作用。我们研究发现,高侵袭特性的HCC细胞来源的外泌体可通过递送其内的转化生长因子(TGF-β)至受体HCC细胞影响其生长微环境,驱动其细胞表型变化,进而影响肿瘤细胞的进展和转移。最后,我们探讨HCC细胞来源的外泌体内其它关键分子如miRNA等在HCC进展、临床诊断和预后判断中的作用。我们应用miRNA芯片方法筛选不同侵袭特性HCC细胞来源的外泌体内显著差异表达的miRNA,根据设定的Cut-off值筛选出miR-665作为候选miRNA,其在HCC中未见报道。研究发现HCC患者血清外泌体miR-665水平较正常对照组明显升高,且与HCC患者肿瘤大小、浸润转移、临床分期以及患者预后等临床病理指标密切相关。进一步体内、外实验研究提示,HCC细胞来源的外泌体可能通过miR-665激活MAPK/ERK通路促进肿瘤细胞的增殖,应用慢病毒转染特异性敲低HCC细胞来源的外泌体miR-665后,效果较前明显减弱。研究提示外泌体内miR-665或可作为HCC诊断及预后判断的有效标志物。此外,我们还对HCC细胞来源的外泌体携带的蛋白进行分析,发现其含有与肿瘤细胞恶性增殖、血管新生密切相关的VEGFR等。综上所述,我们的研究系统探讨了不同侵袭特性HCC细胞来源的外泌体在HCC索拉菲尼抵抗、侵袭转移以及临床诊断、预后判断中的作用和机制。我们发现,HCC细胞来源的外泌体可能通过转运其内的多种生物分子(细胞因子、蛋白、miRNA等)影响受体细胞增殖、转移和药物抵抗等。且HCC细胞来源的外泌体根据其来源细胞侵袭特性不同,其内成分存在表达差异。我们的研究揭示了 HCC来源的外泌体在肿瘤药物抵抗和侵袭转移中的作用和分子机制,对于开发针对外泌体及其内生物分子的新型HCC诊疗手段提供帮助和理论支持。
[Abstract]:Hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) is one of the most common malignant tumors, ranked second range of tumor death associated with prognosis in patients with.HCC. The reason mainly lies in the clinical diagnosis of patients with the disease when period is late, the tumor appeared in liver or distant organ metastasis. Sola Fini is currently the only approved for multiple target drug treated HCC. Sola Feeney resistance is also the main problem affecting the survival of HCC patients. Previous studies found that exosomes as many different types of cells secreting vesicles, can transport a variety of biologically active molecules to target cells, is involved in regulation of tumor microenvironment, thereby affecting tumor progression, metastasis and drug resistance. In addition, outside urinary body from tumor cells carrying its source cell specific proteins, lipids and miRNA, thus the exosomes as a diagnostic biomarker of tumor Material has attracted more and more attention. However, the exosomes on different invasiveness of HCC cells derived from HCC in sorafenib resistant and EMT induced tumor metastasis effect has not been reported. In addition, exosomes key miRNA reported specific role in the diagnosis of HCC and the development of the tumor and the mechanism in this study is less. Exosomes derived from HCC cells as the research focus, to explore the resistance of exosomes derived from HCC cells with different invasive characteristics in HCC Sola Feeney, the potential function and the molecular mechanism of invasion and metastasis and clinical diagnosis. We first extracted by ultracentrifugation method with different invasive characteristics of HCC cell culture supernatant in exosomes, and Application TEM, western, blot and other methods of analysis, identification. Then co cultured with exosomes HCC cell receptor and Sola Feeney and different invasion of HCC cells, HCC cells to investigate the source The relationship between exosomes and tumor cell Sola Feeney resistance. We found that exosomes derived from HCC cells with different invasive properties in vivo and in vitro can induce HCC cell apoptosis resistance Sola Feeney, exosomes and secreted high invasive HCC cells derived from.HCC cells derived from the body more likely to be caused by reverse Sola Feeney and activation HGF/c-Met/Akt pathway, multi drug resistance associated protein up to play this role. Then, we study the invasion of exosomes derived from HCC cells with different invasive characteristics in tumor cells, the effect and mechanism of metastasis. We by cell scratch, exosomes experimental study Transwell different invasiveness of HCC cells on the migration of receptor cells the invasion ability and influence, the cell phenotype protein. And further through the application of exogenous TGF- inhibitor and beta cells treated by LY2109761 extraction Exosomes. We verify the above findings, the high invasiveness of exosomes derived from HCC cells by transformation in the delivery of growth factors (TGF- beta) to HCC cell receptor influences the growth of micro environment, drive the cell phenotype change, thereby affecting the development and metastasis of tumor cells. Finally, we study progress of HCC cells in vivo excretion of other key molecules such as miRNA in HCC, the clinical diagnosis and prognosis of the role. The expression of secreted in vivo significantly we use miRNA chip screening method different invasion of HCC cells derived from miRNA, according to the set value of Cut-off miR-665 is selected as the candidate miRNA in the HCC there was no report. The study found that serum HCC exosome miR-665 level was obviously higher than normal control group, and HCC of patients with tumor size, metastasis, clinical stage and prognosis of patients with clinical pathology etc. The index is closely related. In vivo experiments, suggest that exosomes derived from HCC cells by miR-665 may activate the MAPK/ERK pathway to promote tumor cell proliferation, exosomes miR-665 transfected with lentivirus specific knockdown of HCC cells after effect significantly weakened. Studies suggest that miR-665 can be secreted in vivo or as an effective marker of HCC diagnosis and prognosis. In addition, we also exosomes on HCC cells carrying protein were analyzed, and found it had a malignant tumor cell proliferation, angiogenesis is closely related to the VEGFR. To sum up, our study system of resistance exosomes with different invasive characteristics of HCC cells in HCC Sola Feeney, invasion and metastasis, clinical diagnosis, effect and mechanism of prognosis. We found that exosomes derived from HCC cells through a variety of biological transport in the branch Sub (cytokines, protein, miRNA) receptor cell proliferation, metastasis and drug resistance. And the exosomes derived from HCC cells according to the characteristics of the different sources of cell invasion, the differential expression of the inner components. Our study reveals the exosomes derived from HCC resistance and the role and molecular mechanism of metastasis the invasion in cancer drug development, for the new HCC treatment means for foreign body and urinary in biological molecules to provide help and support the theory.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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