肿瘤微环境在NRP1诱导的肺癌细胞辐射抗性中的作用机制

发布时间：2018-02-02 08:08

本文关键词： 肿瘤微环境 NRP1 辐射抗性三维培养　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：现今,肺癌在全球是排在第一位的恶性肿瘤,对其治疗主要通过手术治疗及放化疗等手段来治愈或控制病情。放疗作为癌症的主要治疗方法,已成为一种治疗肺癌行之有效的方法。但部分病人在接受先进的放疗后,也会产生一定的辐射抗性及随后发生癌症的复发和转移。因此,肿瘤细胞的辐射抗性仍然是肿瘤放疗过程中一个主要障碍。随着细胞生物学技术的发展,人们开始逐渐认识到细胞的增殖、分化、凋亡等一系列生命活动均受到细胞外微环境的影响。而肿瘤微环境又是一个复杂的系统,它由多种基质细胞和细胞因子等组成,期间通过介导复杂的信号通路,使多种炎性因子、趋化因子和血管生成因子等得以分泌,进而加快肿瘤的发展进程。其中神经纤毛蛋白1(neuropilin 1,NRP1)不但与肿瘤的恶性程度相关,同时还可以增强肿瘤的辐射抗性,它可以通过与血管内皮生长因子(VEGF)、信号素(SEMA)和其他多种因子间相互作用,进一步对肿瘤细胞的辐射抗性产生影响。本研究通过在体外构建NRP1表达量不同的A549细胞系,采用三维细胞培养方式建立共培养体系,并利用免疫荧光法、Western blot法、CBA法和流式细胞术等实验技术,探讨肿瘤微环境在NRP1诱导肺癌细胞辐射抗性中的作用机制。首先,通过三维细胞培养技术构建三维细胞模型并对相关标志蛋白进行验证,再利用Jurkat细胞和HLF-1细胞分别与三种A549细胞系进行三维共培养,构建肿瘤炎性微环境及迁移微环境。施加10 Gy的X射线照射后,采用CBA法对肿瘤微环境中相关细胞因子和趋化因子进行检测。结果显示,NRP1可通过对肿瘤微环境中部分炎性因子——TNF、IL-10、IL-8和IL-6,以及部分相关趋化因子——MCP-1、IP-10、RANTES等分泌产生影响,进而诱导肺癌细胞产生辐射抗性作用。最后,采用NRP1低表达细胞模型和A549辐射抗性细胞模型(NRP1高表达),建立与相应细胞系(Jurkat细胞和HLF-1细胞)二维共培养方式,进一步验证了NRP1通过改变肺癌微环境中部分炎性因子、趋化因子及相关信号通路关键因子的分泌,相关细胞组份转化,因而对肺癌细胞的免疫耐受和迁移能力等方面发挥重要作用。因此,本实验主要通过构建相应细胞的二维及三维共培养模型,探讨肺癌的炎性微环境和迁移微环境在NRP1诱导的肺癌细胞辐射抗性中的作用。旨在为临床肺癌的放射治疗提供新的理论基础及实验依据。
[Abstract]:Nowadays, lung cancer is the first malignant tumor in the world. The treatment of lung cancer is mainly through surgical treatment and radiotherapy and chemotherapy to cure or control the disease. Radiotherapy is the main treatment of cancer. It has become an effective method for the treatment of lung cancer. However, after receiving advanced radiotherapy, some patients will also have certain radiation resistance and the recurrence and metastasis of cancer. Radiation resistance of tumor cells is still a major obstacle during radiotherapy. With the development of cell biology, people begin to realize the proliferation and differentiation of tumor cells. Apoptosis and a series of life activities are affected by the extracellular microenvironment. Tumor microenvironment is a complex system, which consists of a variety of stromal cells and cytokines, during which complex signal pathways are mediated. Many inflammatory factors, chemokines and angiogenic factors can be secreted, thus speeding up the development of the tumor, in which neurocilia protein 1 neuropilin 1. NRP1) not only correlates with the malignancy of the tumor, but also enhances the radioresistance of the tumor, which can be mediated by vascular endothelial growth factor (VEGF). Sema) and other factors further affect the radiation resistance of tumor cells. In this study, A549 cell lines with different NRP1 expression levels were constructed in vitro. The co-culture system was established by using three-dimensional cell culture method. Western blot assay and flow cytometry were used. To investigate the role of tumor microenvironment in radioresistance induced by NRP1 in lung cancer cells. Firstly, the three-dimensional cell model was constructed by three-dimensional cell culture and the related marker proteins were verified. Jurkat cells and HLF-1 cells were co-cultured with three kinds of A549 cells respectively to construct inflammatory microenvironment and migration microenvironment. CBA method was used to detect the cytokines and chemokines in tumor microenvironment. The results showed that NRP1 could be used to detect some inflammatory cytokines TNF-IL-10 in tumor microenvironment. The secretion of IL-8 and IL-6, as well as some related chemokines, MCP-1, IP-10, RANTES, and so on, can induce radioresistance in lung cancer cells. Finally. Using NRP1 low expression cell model and A549 radiation resistant cell model, a two dimensional co-culture method was established with the corresponding cell lines: Jurkat cells and HLF-1 cells. It is further verified that NRP1 can change the secretion of inflammatory cytokines, chemokines and key factors related to signaling pathway in the microenvironment of lung cancer, and the transformation of related cell components. Therefore, it plays an important role in the immune tolerance and migration ability of lung cancer cells. Therefore, this experiment mainly through the establishment of the corresponding cell two-dimensional and three-dimensional co-culture model. To explore the role of inflammatory microenvironment and migration microenvironment in radioresistance of lung cancer cells induced by NRP1 in order to provide a new theoretical and experimental basis for radiotherapy of lung cancer.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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