ATM相关信号通路在低剂量辐射诱导肺正常及肿瘤细胞生物学效应差异中的作用及机制研究

发布时间：2018-06-17 07:08

本文选题：低剂量辐射 + 正常肺上皮细胞　；参考：《吉林大学》2016年博士论文

【摘要】：目的:本研究以人肺上皮细胞HBE135-E6E7(HBE)及人肺腺癌细胞A549为研究对象,探讨ATM相关信号通路在LDR诱导这两种细胞生物学效应差异中的作用及相关分子机制。方法:1.通过WST-1及平板克隆形成实验检测HBE及A549两种细胞在LDR作用前后生存活性的变化。2.通过PI单染流式细胞术检测HBE及A549两种细胞在LDR作用前后细胞周期分布变化情况。3.通过Annexin V-FITC/PI双染和JC-1染色流式细胞术检测HBE及A549两种细胞在LDR序贯HDR处理后存活率和线粒体膜电位变化情况;同时通过DCFH-DA探针检测各组细胞内ROS水平变化。4.通过Real-time q PCR方法检测HBE及A549两种细胞在LDR作用前后抗氧化物NQO1及HO-1表达变化。5.通过Western blot方法检测HBE及A549两种细胞在LDR作用前后ATM/p ATM、AKT/p AKT、GSK-3β/p GSK-3β、CDK4、CDK6、cyclin D1、Nrf2、NQO1及HO-1的表达变化。6.通过ATM si RNA转染或caffeine处理明确ATM相关信号通路在LDR诱导HBE及A549细胞产生生物学效应差异中的作用。结果:1.LDR能够诱导HBE细胞产生兴奋性效应,而在A549细胞中无此效应在剂量效应研究中,我们发现:20-100 m Gy的X线能够明显刺激HBE细胞增殖,其中75 m Gy刺激增殖的效应最为明显;然而,在20-200 m Gy的剂量范围内,A549细胞的增殖率并没有明显改变。而当辐射剂量超过200 m Gy时,两种细胞的增殖均受到显著的抑制。在LDR的时间效应研究中,HBE细胞在75 m Gy照射后24-72 h较对照组增殖率明显增加,而A549细胞在LDR后这些时间点增殖率无明显变化。进一步应用平板克隆形成实验发现:HBE细胞在LDR后10天集落形成数量较对照组明显增多,而A549细胞在LDR前后集落形成数量无明显变化。应用流式细胞术检测了75 m Gy X线照射后24 h时两种细胞在各细胞周期中的分布比例。结果显示:HBE细胞在LDR后24 h时S期细胞较对照组比例升高了1.67倍,同时伴随G0/G1期细胞比例下降了1.32倍。相比之下,我们并没有观察到A549细胞在LDR前后各周期细胞比例有明显变化。2.LDR能够诱导HBE细胞产生适应性反应,而在A549细胞中无此效应将细胞分成4组:对照组(假照组),D1(75 m Gy),D2(5 Gy)及D1+D2(75m Gy+5Gy)。应用DCFH-DA探针检测各组细胞内ROS水平变化情况。结果显示:与对照组相比,D2组HBE细胞及A549细胞内ROS水平显著升高。然而,当预先给予D1处理时,在HBE细胞中,D1+D2组ROS水平较D2组明显下降;而在A549细胞中,D1+D2组ROS水平较D2组无明显变化。进一步应用JC-1探针检测4组细胞线粒体膜电位的变化,以及流式细胞术检测4组细胞的凋亡变化情况。结果显示:与对照组相比,D2组HBE及A549细胞线粒体膜电位明显下降。然而,当预先给予D1处理时,在HBE细胞中,D1+D2组线粒体膜电位水平较D2组显著升高;而在A549细胞中,D1+D2组线粒体膜电位水平较D2组无明显变化。流式细胞术检测各组细胞凋亡变化情况与线粒体膜电位变化趋势一致3.ATM及AKT参与LDR诱导HBE细胞兴奋性效应的产生,而在A549细胞中无此效应应用western blot方法检测了ATM及其下游分子AKT的磷酸化水平,结果显示:与对照组相比,HBE细胞在75 m Gy X线照射后24 h时ATM及AKT的磷酸化水平显著升高,但ATM及ATK总蛋白水平无明显变化。然而,在A549细胞中,ATM及AKT蛋白磷酸化水平及总蛋白水平均未受到LDR的影响。应用caffeine或ATM si RNA在LDR前预先处理细胞后,我们发现在HBE细胞中,caffeine或ATM si RNA的加入能够抑制LDR诱导的ATM活化及AKT磷酸化表达;而在A549细胞中,无论是否应用caffeine或ATM si RNA处理,LDR均未影响ATM及AKT的磷酸化水平。随后,我们应用WST-1法检测了caffeine及ATM si RNA处理前后LDR对HBE及A549细胞增殖率的影响。结果显示:在HBE细胞中,加入caffeine或转染ATM si RNA都能够消除LDR对细胞的促增殖效应;而在A549细胞中,加入caffeine或转染ATM si RNA都没有改变细胞的增殖率。4.LDR通过激活ATM/AKT/GSK-3β通路促进HBE细胞内CDK4/CDK6/cyclin D1表达,而在A549细胞中无此效应应用western blot方法检测了LDR后24 h时CDK4,CDK6及cyclin D1的表达变化,同时也检测了AKT及GSK-3β磷酸化水平的变化情况。在HBE细胞中,GSK-3β磷酸化水平、CDK4、CDK6及cyclin D1的表达在LDR后明显升高;应用AKT抑制剂LY294002处理细胞抑制AKT的功能时,LDR诱导GSK-3β磷酸化水平、CDK4、CDK6及cyclin D1的表达水平升高的效应被明显削弱。在A549细胞中,无论是否预先给予LY294002处理细胞,LDR对GSK-3β磷酸化水平、CDK4、CDK6及cyclin D1的表达水平均无明显影响。5.LDR能够诱导HBE细胞Nrf2核积聚,调节抗氧化物m RNA表达,而在A549细胞中无此效应应用实时定量PCR方法检测LDR后Nrf2下游调节的抗氧化物NQO1及HO-1在m RNA水平的表达变化。我们发现,在HBE细胞中,NQO1及HO-1 m RNA表达水平在LDR组较对照组明显升高;而在A549细胞中,NQO1及HO-1 m RNA表达水平在LDR前后均无明显变化。应用caffeine及ATM si MRA预先处理两种细胞后我们发现,预先应用caffeine或ATM si RNA处理细胞都会削弱LDR在HBE细胞中诱导NQO1及HO-1 m RNA表达升高的效应;而在A549细胞中,无上述效应发生。6.LDR通过ATM/AKT/GSK-3β通路激活HBE细胞抗氧化反应,而在A549细胞中无此效应应用western blot方法检测LDR后24 h时细胞内ATM、AKT、GSK-3β磷酸化水平的表达变化,胞浆、胞核内Nrf2蛋白以及NQO1、HO-1蛋白的表达变化。结果显示:在HBE细胞中,与对照组相比,LDR组细胞胞核内Nrf2蛋白及其调节的抗氧化物NQO1及HO-1表达明显升高,这与ATM、AKT、GSK-3β磷酸化水平变化的趋势一致;而在A549细胞中,上述蛋白表达水平均未受到LDR的影响。应用caffeine预先处理细胞,或ATM si RNA预先转染细胞,我们发现:在HBE细胞中,caffeine及ATM si RNA处理均可以抑制LDR诱导胞核Nrf2蛋白及NQO1及HO-1表达升高的效应,且这一结果与ATM、AKT、GSK-3β磷酸化水平变化的趋势一致;而在A549细胞中,我们并没有观察到上述效应的发生。为了验证ATM在LDR诱导HBE细胞产生保护效应中的作用,我们应用ATM si RNA转染细胞24 h后,给予75 m Gy X线照射后24时序贯5 Gy X线照射处理,在照射后24 h后利用WST-1方法检测各组细胞增殖率的变化情况。结果显示,在HBE细胞中,预先应用ATM si RNA转染细胞,能够显著抑制LDR对序贯HDR杀伤细胞的保护效应。结论:1.LDR能够诱导正常肺上皮细胞产生兴奋性效应及适应性反应,而在肺腺癌细胞中没有诱导上述效应的发生。2.LDR诱导生物学效应的差异与ATM相关的促增殖信号通路及抗氧化反应机制相关。在正常细胞中,LDR能够激活ATM蛋白是其诱导兴奋效应发生的始发事件。3.在正常细胞内,由LDR活化的ATM蛋白能够通过激活其下游的AKT/GSK-3β信号通路促进CDK4/CDK6/cyclin D1的表达,诱导正常细胞兴奋性效应的发生,而在肿瘤细胞中无此效应。4.在正常细胞内,由LDR激活的ATM/AKT/GSK-3β信号通路也可以促进Nrf2向细胞核内转移,增加其下游抗氧化物NQO1及HO-1的表达,诱导适应性反应的发生,而在肿瘤细胞中无此效应。本研究基于LDR的生物学效应,探讨了一种LDR序贯HDR的肿瘤治疗新模式的可行性,以期提高肿瘤放疗剂量,进而提高疗效,同时又能减轻正常组织的损伤。LDR对肿瘤细胞是否存在兴奋性效应和适应性反应是LDR临床应用的关键环节。本研究发现LDR对肿瘤细胞及正常细胞产生的生物学效应存在差异,并从细胞增殖、细胞周期、细胞信号传导通路、抗氧化应激反应等方面进行了LDR生物学效应差异机制的对比研究。研究成果将为LDR的临床应用奠定理论及实验基础。
[Abstract]:Objective: the purpose of this study was to investigate the role of ATM related signaling pathway in the differentiation of these two cell biological effects and the related molecular mechanisms of human lung epithelial cell HBE135-E6E7 (HBE) and human lung adenocarcinoma cell A549. Methods: 1. through the formation of WST-1 and flat clones, two cells of HBE and A549 were tested before the action of LDR. The changes of the viability of.2. by PI single dye flow cytometry were used to detect the changes of cell cycle distribution of HBE and A549 two cells before and after the action of LDR.3. through Annexin V-FITC/PI double staining and JC-1 staining flow cytometry to detect the survival rate and the change of the membrane potential of HBE and A549 two cells after LDR sequential treatment. Detection of ROS level changes in each cell by A probe.4. through Real-time Q PCR method to detect HBE and A549 two kinds of cells before and after the action of LDR NQO1 and HO-1. The expression of -1 changes.6. through ATM Si RNA transfection or caffeine treatment to clarify the role of ATM related signaling pathway in the biological effects of LDR induced HBE and A549 cells. The proliferation of HBE cells was significantly stimulated, and the proliferation effect of 75 m Gy stimulation was most obvious; however, the proliferation rate of A549 cells was not significantly changed in the dose range of 20-200 m Gy. The proliferation of two cells was significantly inhibited when the radiation dose exceeded 200 m Gy. In the time effect study of LDR, HBE cells were in 75 m Gy. The proliferation rate of 24-72 h was significantly higher than that of the control group, but the proliferation rate of A549 cells at these time points was not significantly changed after LDR. Further application of flat clones found that the number of colony formation in HBE cells increased significantly at the 10 day after LDR, but the number of colony formation in A549 cells before and after LDR was not significantly changed. The distribution ratio of two cells in each cell cycle at 24 h after 75 m Gy X ray irradiation was measured. The results showed that the proportion of S phase cells in HBE cells was 1.67 times higher than that of the control group at 24 h after LDR, and the proportion of cells in G0/G1 phase decreased by 1.32 times. .2.LDR could induce HBE cells to induce adaptive response, but in A549 cells, the cells were divided into 4 groups without this effect: the control group (false group), D1 (75 m Gy), D2 (5 Gy) and D1+D2 (75m Gy+5Gy). The level of intracellular ROS increased significantly. However, when D1 was given in advance, the level of ROS in group D1+D2 decreased significantly in HBE cells, but in A549 cells, the ROS level of D1+D2 group was not significantly higher than that in the D2 group. Further, JC-1 probe was used to detect the changes in the mitochondrial membrane potential of 4 groups of cells, and the changes of apoptosis in 4 groups of cells were detected by flow cytometry. The results showed that the mitochondrial membrane potential of HBE and A549 cells in the D2 group decreased significantly compared with the control group. However, the mitochondrial membrane potential level in the D1+D2 group was significantly higher than that in the D2 group when the D1 was given in advance, while in A549 cells, the mitochondrial membrane potential level of the D1+D2 group was not significantly higher than that in the D2 group. Flow cytometry was used to detect each group in A549 cells. The changes of apoptosis and mitochondrial membrane potential are consistent with the trend of mitochondrial membrane potential change, 3.ATM and AKT participate in the production of excitatory effects induced by LDR in HBE cells, while Western blot method is used in A549 cells to detect the phosphorylation level of AKT in ATM and its downstream molecules. The results show that, compared with those in the group, the HBE cells are 24 after 75 m Gy. The phosphorylation level of ATM and AKT increased significantly, but there was no significant change in the total protein level of ATM and ATK. However, the phosphorylation level and total protein level of ATM and AKT proteins were not affected by LDR in A549 cells. LDR induced ATM activation and AKT phosphorylation were inhibited, but in A549 cells, no matter whether caffeine or ATM Si RNA treated, LDR did not affect the phosphorylation level of ATM and AKT. The addition of caffeine or transfection of ATM Si RNA can eliminate the proliferation effect of LDR on cells, but in A549 cells, the addition of caffeine or ATM Si RNA does not change the proliferation rate of the cells. The OT method detected the expression of CDK4, CDK6 and cyclin D1 at 24 h after LDR, and also detected the changes in the level of AKT and GSK-3 beta phosphorylation. The effects of elevated levels of acidification, CDK4, CDK6 and cyclin D1 were significantly weakened. In A549 cells, LDR has no significant effects on the level of GSK-3 beta phosphorylation, CDK4, CDK6, and cyclin D1, regardless of whether or not it is pre given to LY294002 cells. We found that the expression of NQO1 and HO-1 at the m RNA level in the Nrf2 downstream of LDR after LDR was detected by real-time quantitative PCR method without this effect. We found that the expression level of NQO1 and HO-1 m in HBE cells was significantly higher than that in the control group in HBE cells. After two cells were pretreated with caffeine and ATM Si MRA, we found that pre application of caffeine or ATM Si RNA processing cells could weaken the effect of LDR in HBE cells to induce NQO1 and HO-1 to increase expression. The changes in the expression of ATM, AKT, GSK-3 beta phosphorylation, cytoplasm, Nrf2 protein and NQO1, HO-1 protein in the cell nucleus were detected in A549 cells without the effect of Western blot in A549 cells. The results showed that in the HBE cells, compared with the control group, the protein and its regulation of oxygen resistance in the cells were compared with the control group. The expression of NQO1 and HO-1 increased obviously, which was in accordance with the trend of ATM, AKT, GSK-3 beta phosphorylation, but in A549 cells, the expression level of these proteins was not affected by LDR. LDR induced the increase in the expression of Nrf2 protein and NQO1 and HO-1, and this result is in accordance with the trend of ATM, AKT, GSK-3 beta phosphorylation. In A549 cells, we did not observe the above effect. In order to verify the effect of ATM on LDR induced HBE cells, we used ATM to transfect cells 2 After 4 h, 75 m Gy X ray irradiation was given at 24 hours after 5 Gy X ray irradiation, and the cell proliferation rate of each group was detected by WST-1 method after 24 h after irradiation. The results showed that the pre application of ATM Si RNA transfected cells in HBE cells could significantly inhibit the protective effect of LDR on the sequential HDR killer cells. There is an exciting and adaptive response in normal lung epithelial cells, but the difference in.2.LDR induced biological effects in the lung adenocarcinoma cells is related to the ATM related proliferation signaling pathway and the mechanism of antioxidant response. In normal cells, LDR can stimulate the activation of ATM protein as the beginning of its induced excitation effect. In normal cells, the ATM protein activated by LDR can promote the expression of CDK4/CDK6/cyclin D1 by activating the AKT/GSK-3 beta signaling pathway in the downstream, inducing the occurrence of the normal cell excitatory effect, while the.4. in the tumor cells is not in the normal cell, and the ATM/AKT/GSK-3 beta signaling pathway activated by LDR can also promote N in the normal cells. The ATM/AKT/GSK-3 beta signaling pathway activated by LDR can also promote N in the normal cells. The ATM activation of the ATM/AKT/GSK-3 beta pathway in the tumor cells can also promote N. The transfer of RF2 into the nucleus increases the expression of the downstream antioxidants, NQO1 and HO-1, and induces the occurrence of adaptive response, but has no effect in the tumor cells. Based on the biological effects of LDR, this study explored the feasibility of a new mode of LDR sequential HDR for tumor treatment, in order to improve the dose of tumor radiotherapy, and then improve the curative effect, and at the same time It is a key link in the clinical application of LDR to reduce the excitatory and adaptive response of.LDR to tumor cells. This study found that the biological effects of LDR on tumor cells and normal cells were different, and from cell proliferation, cell cycle, cell signal transduction pathway, antioxidant stress response and so on. A comparative study on the mechanism of LDR biological effects was conducted. The research results will lay a theoretical and experimental foundation for the clinical application of LDR.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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