Division of Hematology, Department of Medicine, University of Patras, Patras, Rio, 26504, Greece.
J Radiat Res. 2012 Sep;53(5):672-85. doi: 10.1093/jrr/rrs019. Epub 2012 Jul 10.
Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34(+) irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34(+) from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis.
辐射通过产生活性氧（ROS）对 DNA 产生直接和间接的影响。由于 ROS 的产生，辐照造血祖细胞（HPC）经历 DNA 链断裂，有利于遗传不稳定性。我们的目的是研究一系列辐射剂量对 HPC 的影响，以及胰岛素样生长因子-1（IGF-1）激活的可能保护机制。在存在或不存在 IGF-1 的情况下，使用流式细胞术检测人 HPC-CD34(+) 在 1、2 和 5 Gy X 射线照射下的液体培养物中 ROS 的产生。通过 Western blot 分析研究锰超氧化物歧化酶（MnSOD）的表达，并通过免疫荧光分析进行可视化。使用以下测定法估计细胞凋亡：Annexin-V 测定法、DNA 降解测定法、BCL-2/BAX mRNA 和蛋白质水平以及 caspase-9 蛋白免疫荧光可视化。研究了辐照 HPC 的活力和克隆形成能力。超氧阴离子自由基在早期和晚期时间点的生成增加，而晚期时间点的过氧化氢生成稳定。IGF-1 的存在进一步增强了辐照后 24 小时 MnSOD 的辐射诱导增加。IGF-1 通过调节 BAX、BCL-2 和 BCL-2/BAX 比值的 m-RNA 和蛋白质表达以及降低 caspase-9 蛋白表达来抑制线粒体介导的细胞凋亡途径。培养物中 IGF-1 的存在也恢复了辐照细胞的集落形成能力和 HPC 的活力。总之，IGF-1 通过增强 MnSOD 的激活并调节线粒体介导的凋亡途径，消除氧化微环境，从而保护 HPC-CD34(+) 免受辐射影响。
