本文選題：低劑量輻射 + 正常肺上皮細胞��； 參考：《吉林大學》2016年博士論文

【摘要】：目的:本研究以人肺上皮細胞HBE135-E6E7(HBE)及人肺腺癌細胞A549為研究對象,探討ATM相關信號通路在LDR誘導這兩種細胞生物學效應差異中的作用及相關分子機制。方法:1.通過WST-1及平板克隆形成實驗檢測HBE及A549兩種細胞在LDR作用前后生存活性的變化。2.通過PI單染流式細胞術檢測HBE及A549兩種細胞在LDR作用前后細胞周期分布變化情況。3.通過Annexin V-FITC/PI雙染和JC-1染色流式細胞術檢測HBE及A549兩種細胞在LDR序貫HDR處理后存活率和線粒體膜電位變化情況;同時通過DCFH-DA探針檢測各組細胞內(nèi)ROS水平變化。4.通過Real-time q PCR方法檢測HBE及A549兩種細胞在LDR作用前后抗氧化物NQO1及HO-1表達變化。5.通過Western blot方法檢測HBE及A549兩種細胞在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的表達變化。6.通過ATM si RNA轉(zhuǎn)染或caffeine處理明確ATM相關信號通路在LDR誘導HBE及A549細胞產(chǎn)生生物學效應差異中的作用。結果:1.LDR能夠誘導HBE細胞產(chǎn)生興奮性效應,而在A549細胞中無此效應在劑量效應研究中,我們發(fā)現(xiàn):20-100 m Gy的X線能夠明顯刺激HBE細胞增殖,其中75 m Gy刺激增殖的效應最為明顯;然而,在20-200 m Gy的劑量范圍內(nèi),A549細胞的增殖率并沒有明顯改變。而當輻射劑量超過200 m Gy時,兩種細胞的增殖均受到顯著的抑制。在LDR的時間效應研究中,HBE細胞在75 m Gy照射后24-72 h較對照組增殖率明顯增加,而A549細胞在LDR后這些時間點增殖率無明顯變化。進一步應用平板克隆形成實驗發(fā)現(xiàn):HBE細胞在LDR后10天集落形成數(shù)量較對照組明顯增多,而A549細胞在LDR前后集落形成數(shù)量無明顯變化。應用流式細胞術檢測了75 m Gy X線照射后24 h時兩種細胞在各細胞周期中的分布比例。結果顯示:HBE細胞在LDR后24 h時S期細胞較對照組比例升高了1.67倍,同時伴隨G0/G1期細胞比例下降了1.32倍。相比之下,我們并沒有觀察到A549細胞在LDR前后各周期細胞比例有明顯變化。2.LDR能夠誘導HBE細胞產(chǎn)生適應性反應,而在A549細胞中無此效應將細胞分成4組:對照組(假照組),D1(75 m Gy),D2(5 Gy)及D1+D2(75m Gy+5Gy)。應用DCFH-DA探針檢測各組細胞內(nèi)ROS水平變化情況。結果顯示:與對照組相比,D2組HBE細胞及A549細胞內(nèi)ROS水平顯著升高。然而,當預先給予D1處理時,在HBE細胞中,D1+D2組ROS水平較D2組明顯下降;而在A549細胞中,D1+D2組ROS水平較D2組無明顯變化。進一步應用JC-1探針檢測4組細胞線粒體膜電位的變化,以及流式細胞術檢測4組細胞的凋亡變化情況。結果顯示:與對照組相比,D2組HBE及A549細胞線粒體膜電位明顯下降。然而,當預先給予D1處理時,在HBE細胞中,D1+D2組線粒體膜電位水平較D2組顯著升高;而在A549細胞中,D1+D2組線粒體膜電位水平較D2組無明顯變化。流式細胞術檢測各組細胞凋亡變化情況與線粒體膜電位變化趨勢一致3.ATM及AKT參與LDR誘導HBE細胞興奮性效應的產(chǎn)生,而在A549細胞中無此效應應用western blot方法檢測了ATM及其下游分子AKT的磷酸化水平,結果顯示:與對照組相比,HBE細胞在75 m Gy X線照射后24 h時ATM及AKT的磷酸化水平顯著升高,但ATM及ATK總蛋白水平無明顯變化。然而,在A549細胞中,ATM及AKT蛋白磷酸化水平及總蛋白水平均未受到LDR的影響。應用caffeine或ATM si RNA在LDR前預先處理細胞后,我們發(fā)現(xiàn)在HBE細胞中,caffeine或ATM si RNA的加入能夠抑制LDR誘導的ATM活化及AKT磷酸化表達;而在A549細胞中,無論是否應用caffeine或ATM si RNA處理,LDR均未影響ATM及AKT的磷酸化水平。隨后,我們應用WST-1法檢測了caffeine及ATM si RNA處理前后LDR對HBE及A549細胞增殖率的影響。結果顯示:在HBE細胞中,加入caffeine或轉(zhuǎn)染ATM si RNA都能夠消除LDR對細胞的促增殖效應;而在A549細胞中,加入caffeine或轉(zhuǎn)染ATM si RNA都沒有改變細胞的增殖率。4.LDR通過激活ATM/AKT/GSK-3β通路促進HBE細胞內(nèi)CDK4/CDK6/cyclin D1表達,而在A549細胞中無此效應應用western blot方法檢測了LDR后24 h時CDK4,CDK6及cyclin D1的表達變化,同時也檢測了AKT及GSK-3β磷酸化水平的變化情況。在HBE細胞中,GSK-3β磷酸化水平、CDK4、CDK6及cyclin D1的表達在LDR后明顯升高;應用AKT抑制劑LY294002處理細胞抑制AKT的功能時,LDR誘導GSK-3β磷酸化水平、CDK4、CDK6及cyclin D1的表達水平升高的效應被明顯削弱。在A549細胞中,無論是否預先給予LY294002處理細胞,LDR對GSK-3β磷酸化水平、CDK4、CDK6及cyclin D1的表達水平均無明顯影響。5.LDR能夠誘導HBE細胞Nrf2核積聚,調(diào)節(jié)抗氧化物m RNA表達,而在A549細胞中無此效應應用實時定量PCR方法檢測LDR后Nrf2下游調(diào)節(jié)的抗氧化物NQO1及HO-1在m RNA水平的表達變化。我們發(fā)現(xiàn),在HBE細胞中,NQO1及HO-1 m RNA表達水平在LDR組較對照組明顯升高;而在A549細胞中,NQO1及HO-1 m RNA表達水平在LDR前后均無明顯變化。應用caffeine及ATM si MRA預先處理兩種細胞后我們發(fā)現(xiàn),預先應用caffeine或ATM si RNA處理細胞都會削弱LDR在HBE細胞中誘導NQO1及HO-1 m RNA表達升高的效應;而在A549細胞中,無上述效應發(fā)生。6.LDR通過ATM/AKT/GSK-3β通路激活HBE細胞抗氧化反應,而在A549細胞中無此效應應用western blot方法檢測LDR后24 h時細胞內(nèi)ATM、AKT、GSK-3β磷酸化水平的表達變化,胞漿、胞核內(nèi)Nrf2蛋白以及NQO1、HO-1蛋白的表達變化。結果顯示:在HBE細胞中,與對照組相比,LDR組細胞胞核內(nèi)Nrf2蛋白及其調(diào)節(jié)的抗氧化物NQO1及HO-1表達明顯升高,這與ATM、AKT、GSK-3β磷酸化水平變化的趨勢一致;而在A549細胞中,上述蛋白表達水平均未受到LDR的影響。應用caffeine預先處理細胞,或ATM si RNA預先轉(zhuǎn)染細胞,我們發(fā)現(xiàn):在HBE細胞中,caffeine及ATM si RNA處理均可以抑制LDR誘導胞核Nrf2蛋白及NQO1及HO-1表達升高的效應,且這一結果與ATM、AKT、GSK-3β磷酸化水平變化的趨勢一致;而在A549細胞中,我們并沒有觀察到上述效應的發(fā)生。為了驗證ATM在LDR誘導HBE細胞產(chǎn)生保護效應中的作用,我們應用ATM si RNA轉(zhuǎn)染細胞24 h后,給予75 m Gy X線照射后24時序貫5 Gy X線照射處理,在照射后24 h后利用WST-1方法檢測各組細胞增殖率的變化情況。結果顯示,在HBE細胞中,預先應用ATM si RNA轉(zhuǎn)染細胞,能夠顯著抑制LDR對序貫HDR殺傷細胞的保護效應。結論:1.LDR能夠誘導正常肺上皮細胞產(chǎn)生興奮性效應及適應性反應,而在肺腺癌細胞中沒有誘導上述效應的發(fā)生。2.LDR誘導生物學效應的差異與ATM相關的促增殖信號通路及抗氧化反應機制相關。在正常細胞中,LDR能夠激活ATM蛋白是其誘導興奮效應發(fā)生的始發(fā)事件。3.在正常細胞內(nèi),由LDR活化的ATM蛋白能夠通過激活其下游的AKT/GSK-3β信號通路促進CDK4/CDK6/cyclin D1的表達,誘導正常細胞興奮性效應的發(fā)生,而在腫瘤細胞中無此效應。4.在正常細胞內(nèi),由LDR激活的ATM/AKT/GSK-3β信號通路也可以促進Nrf2向細胞核內(nèi)轉(zhuǎn)移,增加其下游抗氧化物NQO1及HO-1的表達,誘導適應性反應的發(fā)生,而在腫瘤細胞中無此效應。本研究基于LDR的生物學效應,探討了一種LDR序貫HDR的腫瘤治療新模式的可行性,以期提高腫瘤放療劑量,進而提高療效,同時又能減輕正常組織的損傷。LDR對腫瘤細胞是否存在興奮性效應和適應性反應是LDR臨床應用的關鍵環(huán)節(jié)。本研究發(fā)現(xiàn)LDR對腫瘤細胞及正常細胞產(chǎn)生的生物學效應存在差異,并從細胞增殖、細胞周期、細胞信號傳導通路、抗氧化應激反應等方面進行了LDR生物學效應差異機制的對比研究。研究成果將為LDR的臨床應用奠定理論及實驗基礎。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2

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