本文關(guān)鍵詞： 腫瘤微環(huán)境 NRP1 輻射抗性 三維培養(yǎng)　出處：《吉林大學》2017年碩士論文　論文類型：學位論文

【摘要】：現(xiàn)今,肺癌在全球是排在第一位的惡性腫瘤,對其治療主要通過手術(shù)治療及放化療等手段來治愈或控制病情。放療作為癌癥的主要治療方法,已成為一種治療肺癌行之有效的方法。但部分病人在接受先進的放療后,也會產(chǎn)生一定的輻射抗性及隨后發(fā)生癌癥的復(fù)發(fā)和轉(zhuǎn)移。因此,腫瘤細胞的輻射抗性仍然是腫瘤放療過程中一個主要障礙。隨著細胞生物學技術(shù)的發(fā)展,人們開始逐漸認識到細胞的增殖、分化、凋亡等一系列生命活動均受到細胞外微環(huán)境的影響。而腫瘤微環(huán)境又是一個復(fù)雜的系統(tǒng),它由多種基質(zhì)細胞和細胞因子等組成,期間通過介導(dǎo)復(fù)雜的信號通路,使多種炎性因子、趨化因子和血管生成因子等得以分泌,進而加快腫瘤的發(fā)展進程。其中神經(jīng)纖毛蛋白1(neuropilin 1,NRP1)不但與腫瘤的惡性程度相關(guān),同時還可以增強腫瘤的輻射抗性,它可以通過與血管內(nèi)皮生長因子(VEGF)、信號素(SEMA)和其他多種因子間相互作用,進一步對腫瘤細胞的輻射抗性產(chǎn)生影響。本研究通過在體外構(gòu)建NRP1表達量不同的A549細胞系,采用三維細胞培養(yǎng)方式建立共培養(yǎng)體系,并利用免疫熒光法、Western blot法、CBA法和流式細胞術(shù)等實驗技術(shù),探討腫瘤微環(huán)境在NRP1誘導(dǎo)肺癌細胞輻射抗性中的作用機制。首先,通過三維細胞培養(yǎng)技術(shù)構(gòu)建三維細胞模型并對相關(guān)標志蛋白進行驗證,再利用Jurkat細胞和HLF-1細胞分別與三種A549細胞系進行三維共培養(yǎng),構(gòu)建腫瘤炎性微環(huán)境及遷移微環(huán)境。施加10 Gy的X射線照射后,采用CBA法對腫瘤微環(huán)境中相關(guān)細胞因子和趨化因子進行檢測。結(jié)果顯示,NRP1可通過對腫瘤微環(huán)境中部分炎性因子——TNF、IL-10、IL-8和IL-6,以及部分相關(guān)趨化因子——MCP-1、IP-10、RANTES等分泌產(chǎn)生影響,進而誘導(dǎo)肺癌細胞產(chǎn)生輻射抗性作用。最后,采用NRP1低表達細胞模型和A549輻射抗性細胞模型(NRP1高表達),建立與相應(yīng)細胞系(Jurkat細胞和HLF-1細胞)二維共培養(yǎng)方式,進一步驗證了NRP1通過改變肺癌微環(huán)境中部分炎性因子、趨化因子及相關(guān)信號通路關(guān)鍵因子的分泌,相關(guān)細胞組份轉(zhuǎn)化,因而對肺癌細胞的免疫耐受和遷移能力等方面發(fā)揮重要作用。因此,本實驗主要通過構(gòu)建相應(yīng)細胞的二維及三維共培養(yǎng)模型,探討肺癌的炎性微環(huán)境和遷移微環(huán)境在NRP1誘導(dǎo)的肺癌細胞輻射抗性中的作用。旨在為臨床肺癌的放射治療提供新的理論基礎(chǔ)及實驗依據(jù)。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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