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放疗被广泛应用于肺癌的临床治疗,但肺癌细胞对射线的敏感性不同,在放疗过程中会发生辐射抵抗,因此比较不同肺癌细胞的辐射抗性,探究提高肺癌细胞辐射敏感性的方法具有重要意义。
非小细胞肺癌(non-small-cell lung cancer,NSCLC)是发病率最高、研究最多的肺癌。过去20多年,虽然研究人员深入了解了NSCLC的分子基础和基因基础,但肺癌患者总的生存率并没有明显提高。肺癌细胞产生放化疗抵抗是临床治疗效果差的重要原因,肺癌细胞抗性产生的机制亟待探究。放疗和一些化疗药物都可以通过升高细胞内活性氧(ROS)的水平造成DNA损伤,从而导致肺癌细胞死亡或抑制其增殖、转移。然而,肺癌细胞内抗氧化系统的相关基因突变和蛋白丰度的变化会导致其产生放化疗抵抗[1-2]。
核因子E2相关因子2(nuclear factor erythroid-2-related factor 2,Nrf2)作为一种核转录因子可以与抗氧化作用元件(ARE)结合调节一系列抗氧化基因的表达,如HO-1(heme oxygenase-1,血红素加氧酶-1)、NQO1(NAD(P)H:quinone oxidoreductase 1,还原型辅酶/醌氧化还原酶1)、GSTs(glutathione S-transferase,谷胱甘肽S-转移酶)[3-5],也可以调节药物的解毒作用和药物泵出[6]。Nrf2在人体各组织器官中广泛表达,它的含量主要受到Keap1的调节[7]。正常情况下,Nrf2在胞质中可与Keap1结合发生泛素化,并被转运到蛋白酶体降解,氧化应激可使二者解聚,游离Nrf2可以稳定存在并转位入细胞核发挥转录因子的作用[5, 8]。肺癌细胞中Nrf2常过表达,且Nrf2过表达的肺癌患者较Nrf2正常表达的患者预后差[9]。研究人员对NSCLC的辐射抗性机制做了大量研究工作,但Nrf2蛋白与NSCLC辐射敏感性的相关报道很少。由于不同类型肺癌肿瘤由不同类型细胞组成,因此临床上对放疗敏感性的表现不同,探究不同肺癌细胞的辐射敏感性差异以及导致这种差异的原因,有助于临床医师针对不同患者制定更精准的治疗方案。A549属于鳞状NSCLC细胞,来源于外植体肺肿瘤,H460属于鳞状大细胞肺癌细胞,来源于胸膜积液,两者在耐药性和辐射抗性等方面存在较大差异,但具体机制还不明确。笔者通过对肺癌A549和H460两个细胞系的辐射敏感性进行比较,并对比两者的Nrf2蛋白含量,探讨Nrf2蛋白水平差异对两种细胞系辐射敏感性的影响。
肺癌细胞A549和H460对137Cs γ射线辐射敏感性差异的研究
Difference of radiosensitivity for 137Cs γ-radiation between A549 and H460 lung cancer cell lines
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摘要:
目的研究肺癌细胞A549和H460对137Cs γ射线的辐射敏感性差异及核因子E2相关因子2(Nrf2)蛋白含量的差异。 方法使用2、4、6 Gy 137Cs γ射线照射A549和H460细胞;1、2、4、6 Gy 137Cs γ射线照射H460细胞,用克隆形成法检测细胞增殖能力,单细胞凝胶电泳检测细胞DNA损伤修复情况,casp_1.2.3b1彗星分析软件分析olive尾距值和尾部DNA含量,蛋白质印迹法检测Nrf2蛋白表达量。克隆形成率、olive尾距值和尾部DNA含量采用独立样本t检验进行比较。 结果经2、4、6 Gy 137Cs γ射线照射后,肺癌A549细胞的克隆形成率分别为(73.78±14.69)%、(42.26±3.19)%、(17.50±2.18)%;H460细胞的克隆形成率分别为(56.38±6.28)%、(23.82±8.25)%、(4.66±0.87)%,肺癌A549细胞克隆形成率高于H460细胞,且差异均有统计学意义(t=7.99,P=0.015;t=6.75,P=0.019;t=12.03,P=0.005)。4 Gy照射后2 h,肺癌H460细胞的olive尾距值(1.27±0.05)和尾部DNA含量(4.51±0.19)%明显高于A549细胞[0.68±0.04、(2.12±0.14)%],且差异均有统计学意义(t=8.69、10.30,均P < 0.05)。蛋白质印迹实验结果显示,肺癌A549比H460细胞系的Nrf2蛋白丰度高,照射后两种细胞中的Nrf2蛋白水平均升高,但肺癌A549细胞明显高于H460细胞。 结论肺癌A549细胞系对137Cs γ射线的辐射抗性强于H460细胞系,这种辐射抗性差异可能与两种细胞系内Nrf2蛋白的含量相关。 Abstract:ObjectiveThis study aims to compare radiosensitivity to γ ray between A549 and H460 cells and explore the relationship between different radiosensitivities and Nrf2 expression. MethodsA549 and H460 cells were exposed to 2, 4, and 6 Gy 137Cs γ ray, and H460 cells were exposed to 1, 2, 4, and 6 Gy 137Cs γ ray. Cell proliferation was assessed by clone formation assay. DNA damage was evaluated using comet assay. Nrf2 protein level was measured by Western blot analysis. ResultsClone formation assay indicated that the clone formation rates of A549 cells were (73.78±14.69)%, (42.26±3.19)%, and (17.5±2.18)%, and those of H460 cells were (56.38±6.28)%, (23.82±8.25)%, and (4.66±0.87)% after exposure to 2, 4, and 6 Gy, respectively (t=7.99, P=0.015; t=6.75, P=0.019; t=12.03, P=0.005). Lung cancer H460 cells possessed higher olive tail moments (1.27±0.05), and tail DNA(4.51±0.19)% than A549 cells[0.68±0.04, (2.12±0.14)%] in the comet assay conducted 2 h after 4 Gy irradiation(t=8.69, 10.30, both P < 0.05). The Western blot assay indicated that the Nrf2 protein level was higher in A549 cells than in H460 cells. Radiation might induce the increase in the Nrf2 protein level in A549 and H460 cells. Moreover, the Nrf2 protein level was higher in radioresistant H460R cells than in radiosensitive H460 cells. ConclusionA549 cells are more resistant to 137Cs γ ray than H460 cells, and different radiosensitivities may be related to Nrf2 protein level. -
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