本文關(guān)鍵詞：Nrf2介導(dǎo)Notch1對非小細(xì)胞肺癌輻射敏感性及侵襲轉(zhuǎn)移的作用研究　出處：《中國科學(xué)院大學(xué)(中國科學(xué)院近代物理研究所)》2017年博士論文　論文類型：學(xué)位論文

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【摘要】：肺癌是全球致死率最高的癌癥,每年因肺癌死亡的人數(shù)已經(jīng)超過結(jié)腸癌、前列腺癌和乳腺癌的總和。肺癌通常分為小細(xì)胞肺癌(SCLC)和非小細(xì)胞肺癌(NSCLC),其中NSCLC占肺癌的85%以上,并且70%的NSCLC肺癌患者被發(fā)現(xiàn)時已是肺癌晚期。由于放射治療的高效性,已被廣泛應(yīng)用于NSCLC的治療中,但電離輻射引起的副作用也不能忽略。射線作用于細(xì)胞,使細(xì)胞產(chǎn)生大量的活性氧自由基(ROS),過多的ROS造成細(xì)胞的凋亡、壞死。NSCLC細(xì)胞擁有比正常肺細(xì)胞更強(qiáng)的抗氧化物酶系統(tǒng),使得其細(xì)胞內(nèi)ROS水平含量較低,這一較強(qiáng)的抗氧化能力使得腫瘤細(xì)胞接收放射治療后存在輻射耐受性。隨著放射治療方式和手段不斷發(fā)展,許多早期肺癌患者都可治愈,但NSCLC的五年生存率只有15%,主要原因是電離輻射促使細(xì)胞侵襲轉(zhuǎn)移的能力增強(qiáng),造成NSCLC的復(fù)發(fā)。目前為止,NSCLC的輻射敏感性及侵襲轉(zhuǎn)移機(jī)制尚不明確,因此,更深層次地研究其機(jī)制對降低放射治療劑量、研制NSCLC靶向藥物意義重大。Nrf2信號通路主要調(diào)控的靶基因是各種抗氧化或解毒蛋白,這些蛋白的激活是機(jī)體抵御氧化應(yīng)激損傷的重要作用因子。Notch1信號通路主要參與機(jī)體的早期發(fā)育、細(xì)胞分化、組織修復(fù)、增殖和凋亡等過程。Nrf2和Notch1在NSCLC中呈異常表達(dá),可影響到細(xì)胞的輻射敏感性和侵襲轉(zhuǎn)移性,其機(jī)制尚不清楚。有研究表明,Nrf2可以調(diào)控Notch1的表達(dá),但這種調(diào)控在NSCLC的輻射響應(yīng)方面卻未見報道。本論文通過蛋白印跡雜交和熒光定量PCR技術(shù)檢測了電離輻射后NSCLC細(xì)胞中Nrf2蛋白及轉(zhuǎn)錄水平變化,確定了電離輻射可激活NSCLC中Nrf2的表達(dá)。通過細(xì)胞免疫熒光、活性氧檢測、細(xì)胞增殖、克隆形成、蛋白印跡雜交和熒光定量PCR等技術(shù)檢測了Nrf2對NSCLC輻射敏感性的影響及Nrf2對Notch1的調(diào)控作用。通過細(xì)胞遷移、侵襲、ELISA酶聯(lián)吸附、蛋白印跡雜交和細(xì)胞免疫熒光等技術(shù)檢測了Nrf2-Notch1信號軸對NSCLC的輻射敏感性和侵襲轉(zhuǎn)移的響。實驗結(jié)果歸納如下:1.X射線可激活A(yù)549細(xì)胞系中Nrf2蛋白的表達(dá),并隨劑量的增加,Nrf2蛋白的表達(dá)也有所增加,并在4 Gy的X射線或2 Gy的碳離子照射24h時Nrf2信號通路處于完全激活狀態(tài)。同時,對Nrf2降解途徑進(jìn)行研究,發(fā)現(xiàn)2μM蛋白酶體抑制劑MG132處理16h,Nrf2降解途徑完全被抑制。2.電離輻射可促進(jìn)Nrf2蛋白的核轉(zhuǎn)位,采用si RNA技術(shù)降低Nrf2的蛋白表達(dá)水平,可降低細(xì)胞輻射誘導(dǎo)的抗氧化物酶的表達(dá),增強(qiáng)ROS含量,降低細(xì)胞增殖,誘導(dǎo)細(xì)胞凋亡。同時,下調(diào)Nrf2也可降低Notch1信號通路的表達(dá),提出基于電離輻射條件下,Nrf2-Notch1信號軸的下降可增強(qiáng)細(xì)胞的輻射敏感性。3.電離輻射可促進(jìn)NSCLC的細(xì)胞遷移和侵襲。下調(diào)Nrf2、Notch1發(fā)現(xiàn)可增強(qiáng)輻射輻射條件下,細(xì)胞E-cadherin蛋白的表達(dá),降低N-cadherin、MMP-2和MMP-9蛋白的表達(dá)。Nrf2-Notch1信號軸的下調(diào)可緩解輻射引起的細(xì)胞侵襲轉(zhuǎn)移的增加。本課題基于電離輻射條件下,NSCLC內(nèi)Nrf2蛋白的變化及相關(guān)信號通路的研究,首次報道了Nrf2可調(diào)控NSCLC內(nèi)Notch1及其下游信號通路的變化,證明了Nrf2及Notch1蛋白可以作為調(diào)節(jié)NSCLC輻射敏感性及侵襲轉(zhuǎn)移的靶基因,為NSCLC的放射治療提供了思路和方法。
[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.

【學(xué)位授予單位】：中國科學(xué)院大學(xué)(中國科學(xué)院近代物理研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R734.2

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