Nrf2介导Notch1对非小细胞肺癌辐射敏感性及侵袭转移的作用研究

发布时间：2018-01-07 06:28

本文关键词：Nrf2介导Notch1对非小细胞肺癌辐射敏感性及侵袭转移的作用研究　出处：《中国科学院大学(中国科学院近代物理研究所)》2017年博士论文　论文类型：学位论文

【摘要】：肺癌是全球致死率最高的癌症,每年因肺癌死亡的人数已经超过结肠癌、前列腺癌和乳腺癌的总和。肺癌通常分为小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC),其中NSCLC占肺癌的85%以上,并且70%的NSCLC肺癌患者被发现时已是肺癌晚期。由于放射治疗的高效性,已被广泛应用于NSCLC的治疗中,但电离辐射引起的副作用也不能忽略。射线作用于细胞,使细胞产生大量的活性氧自由基(ROS),过多的ROS造成细胞的凋亡、坏死。NSCLC细胞拥有比正常肺细胞更强的抗氧化物酶系统,使得其细胞内ROS水平含量较低,这一较强的抗氧化能力使得肿瘤细胞接收放射治疗后存在辐射耐受性。随着放射治疗方式和手段不断发展,许多早期肺癌患者都可治愈,但NSCLC的五年生存率只有15%,主要原因是电离辐射促使细胞侵袭转移的能力增强,造成NSCLC的复发。目前为止,NSCLC的辐射敏感性及侵袭转移机制尚不明确,因此,更深层次地研究其机制对降低放射治疗剂量、研制NSCLC靶向药物意义重大。Nrf2信号通路主要调控的靶基因是各种抗氧化或解毒蛋白,这些蛋白的激活是机体抵御氧化应激损伤的重要作用因子。Notch1信号通路主要参与机体的早期发育、细胞分化、组织修复、增殖和凋亡等过程。Nrf2和Notch1在NSCLC中呈异常表达,可影响到细胞的辐射敏感性和侵袭转移性,其机制尚不清楚。有研究表明,Nrf2可以调控Notch1的表达,但这种调控在NSCLC的辐射响应方面却未见报道。本论文通过蛋白印迹杂交和荧光定量PCR技术检测了电离辐射后NSCLC细胞中Nrf2蛋白及转录水平变化,确定了电离辐射可激活NSCLC中Nrf2的表达。通过细胞免疫荧光、活性氧检测、细胞增殖、克隆形成、蛋白印迹杂交和荧光定量PCR等技术检测了Nrf2对NSCLC辐射敏感性的影响及Nrf2对Notch1的调控作用。通过细胞迁移、侵袭、ELISA酶联吸附、蛋白印迹杂交和细胞免疫荧光等技术检测了Nrf2-Notch1信号轴对NSCLC的辐射敏感性和侵袭转移的响。实验结果归纳如下:1.X射线可激活A549细胞系中Nrf2蛋白的表达,并随剂量的增加,Nrf2蛋白的表达也有所增加,并在4 Gy的X射线或2 Gy的碳离子照射24h时Nrf2信号通路处于完全激活状态。同时,对Nrf2降解途径进行研究,发现2μM蛋白酶体抑制剂MG132处理16h,Nrf2降解途径完全被抑制。2.电离辐射可促进Nrf2蛋白的核转位,采用si RNA技术降低Nrf2的蛋白表达水平,可降低细胞辐射诱导的抗氧化物酶的表达,增强ROS含量,降低细胞增殖,诱导细胞凋亡。同时,下调Nrf2也可降低Notch1信号通路的表达,提出基于电离辐射条件下,Nrf2-Notch1信号轴的下降可增强细胞的辐射敏感性。3.电离辐射可促进NSCLC的细胞迁移和侵袭。下调Nrf2、Notch1发现可增强辐射辐射条件下,细胞E-cadherin蛋白的表达,降低N-cadherin、MMP-2和MMP-9蛋白的表达。Nrf2-Notch1信号轴的下调可缓解辐射引起的细胞侵袭转移的增加。本课题基于电离辐射条件下,NSCLC内Nrf2蛋白的变化及相关信号通路的研究,首次报道了Nrf2可调控NSCLC内Notch1及其下游信号通路的变化,证明了Nrf2及Notch1蛋白可以作为调节NSCLC辐射敏感性及侵袭转移的靶基因,为NSCLC的放射治疗提供了思路和方法。
[Abstract]:Lung cancer is the world's highest death rate of cancer, the annual number of deaths from lung cancer has more than the sum of colon cancer, prostate cancer and breast cancer. Lung cancer is usually divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which accounted for 85% of the above NSCLC lung cancer, and 70% lung cancer patients with NSCLC was found is lung cancer late. Because the efficiency of radiation therapy, has been widely used in the treatment of NSCLC, but the side effects caused by ionizing radiation can not be ignored. Ray effects on the cells, the cells produce free radicals (ROS), caused by excessive ROS cell apoptosis, cell necrosis with.NSCLC ratio of antioxidant enzyme system in normal lung cells, the ROS levels in the cells is low, the strong antioxidant ability of tumor cells are receiving radiation tolerance after radiotherapy with radiotherapy. Methods and means The continuous development of many early lung cancer patients can be cured, but NSCLC five year survival rate is only 15%, the main reason is to enhance the ability of ionizing radiation that promote cell invasion and metastasis, resulting in the recurrence of NSCLC. So far, NSCLC radiation sensitivity and mechanism of invasion and metastasis is not clear, therefore, a deeper study on its mechanism to reduce the radiation dose, the target gene developed drugs targeting NSCLC significant.Nrf2 signal pathway regulation is the main antioxidant or detoxification proteins, these proteins are activated in early stage of development, the body against oxidative stress the important role of the.Notch1 pathway is mainly involved in cell differentiation, tissue repair, proliferation and apoptosis of.Nrf2 and etc. Notch1 showed abnormal expression in NSCLC, radiation sensitivity and can affect the cell invasion and metastasis, the mechanism is still unclear. Studies have shown that Nrf2 can regulate No The expression of tch1, but this regulation has not been reported in response to NSCLC radiation. The detected changes of Nrf2 protein and mRNA in NSCLC cells after ionizing radiation by Western blot and fluorescence quantitative PCR technology, the ionizing radiation can activate NSCLC in Nrf2 expression. By immunofluorescence, active oxygen detection cell proliferation, clone formation, western blot and fluorescence quantitative PCR technique to detect the regulatory role of Nrf2 in radiation sensitivity effect on NSCLC and Nrf2 to Notch1. The cell migration, invasion, ELISA enzyme linkedimmunosorbent, protein blotting and immunofluorescence technique to detect the Nrf2-Notch1 signal axis of NSCLC radiation sensitivity and the invasion and metastasis of rings. The experimental results are as follows: the expression of 1.X - ray activation of Nrf2 protein in A549 cell line, and with the increase of dose, the expression of Nrf2 protein has also been increasing, And Nrf2 in carbon ion irradiation of 24h X ray 4 Gy or 2 Gy when the signal pathway is fully activated. At the same time, the research on Nrf2 degradation pathway, found that 2 M proteasome inhibitor MG132 16h Nrf2 degradation pathway was completely inhibited.2. ionizing radiation can promote the nuclear translocation of Nrf2 protein by Si RNA. Technology to reduce Nrf2 protein expression, expression of antioxidant enzymes can reduce cells induced by radiation, increasing the content of ROS, reduce the cell proliferation and induce cell apoptosis. At the same time, the down-regulation of Nrf2 may reduce the expression of Notch1 signaling pathway, based on the radiation conditions, the decrease in the Nrf2-Notch1 signal axis can enhance the radiation sensitivity of.3. radiation cells can promote cell migration and invasion of NSCLC. The down-regulation of Nrf2, Notch1 found the radiation condition, the expression of E-cadherin protein in cells, decreased N-cadherin, MMP-2 and MMP-9 protein expression .Nrf2-Notch1 signal axis down can alleviate radiation-induced cell invasion and metastasis. This project is based on the radiation conditions, changes of Nrf2 protein in NSCLC and related signal pathway, was reported for the first time Nrf2 can change the regulation of NSCLC in Notch1 and its downstream signaling pathway, proved that Nrf2 and Notch1 protein can be used as target gene regulation of NSCLC radiation sensitivity of invasion and metastasis, to provide ideas and methods for the therapy of NSCLC.

【学位授予单位】：中国科学院大学(中国科学院近代物理研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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