Department of Radiation Oncology, UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania 15232.
Department of Biostatistics, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
Radiat Res. 2021 Sep 1;196(3):235-249. doi: 10.1667/RADE-20-00286.1.
We defined the time course of ionizing radiation-induced senescence in lung compared to bone marrow of p16+/LUC mice in which the senescence-induced biomarker (p16) is linked to a luciferase reporter gene. Periodic in situ imaging revealed increased luciferase activity in the lungs of 20 Gy thoracic irradiated, but not 8 Gy total-body irradiated (TBI) mice beginning at day 75 and increasing to day 170. In serial sections of explanted lungs, senescent cells appeared in the same areas as did fibrosis in the 20 Gy thoracic irradiated, but not the 8 Gy TBI group. Lungs from 8 Gy TBI mice at one year did show increased RNA levels for p16, p21, p19 and TGF-β. Individual senescent cells in 20 Gy irradiated mouse lung included those with epithelial, endothelial, fibroblast and hematopoietic cell biomarkers. Rare senescent cells in the lungs of 8 Gy TBI mice at one year were of endothelial phenotype. Long-term bone marrow cultures (LTBMCs) were established at either day 60 or one year after 8 Gy TBI. In freshly removed marrow at both times after irradiation, there were increased senescent cells. In LTBMCs, there were increased senescent cells in both weekly harvested single cells and in colonies of multilineage hematopoietic progenitor cells producing CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, mega-karyocyte) that were formed in secondary cultures when these single cells were plated in semisolid media. LTBMCs from TBI mice produced fewer CFU-GEMM; however, the relative percentage of senescent cell-containing colonies was increased as measured by both p16-luciferase and β-galactosidase. Therefore, 20 Gy thoracic radiation, as well as 8 Gy TBI, induces senescent cells in the lungs. With bone marrow, 8 Gy TBI induced senescence in both hematopoietic cells and in colony-forming progenitors. The p16+/LUC mouse strain provides a valuable system in which to compare the kinetics of radiation-induced senescence between organs in vivo, and to evaluate the potential role of senescent cells in irradiation pulmonary fibrosis.
我们定义了 p16+/LUC 小鼠肺部与骨髓中电离辐射诱导衰老的时间进程，其中衰老诱导生物标志物（p16）与荧光素酶报告基因相连。定期原位成像显示，20 Gy 胸部照射但 8 Gy 全身照射（TBI）的 p16+/LUC 小鼠肺部的荧光素酶活性从第 75 天开始增加，并持续增加到第 170 天。在离体肺的连续切片中，在 20 Gy 胸部照射组中，与纤维化出现在相同区域的衰老细胞，但在 8 Gy TBI 组中没有出现。一年后，8 Gy TBI 小鼠的肺部显示出 p16、p21、p19 和 TGF-β 的 RNA 水平增加。20 Gy 照射的小鼠肺部中的单个衰老细胞包括具有上皮细胞、内皮细胞、成纤维细胞和造血细胞生物标志物的细胞。一年后，8 Gy TBI 小鼠肺部罕见的衰老细胞表现为内皮表型。8 Gy TBI 后第 60 天或一年后建立长期骨髓培养物（LTBMC）。在照射后两个时间点的新鲜取出的骨髓中，都有更多的衰老细胞。在 LTBMC 中，每周收获的单个细胞和多谱系造血祖细胞形成的 CFU-GEMM（集落形成单位-粒细胞、红细胞、单核细胞/巨噬细胞、巨核细胞）的集落中都有更多的衰老细胞，这些单个细胞在半固体培养基中接种时，在次级培养中形成 CFU-GEMM 集落。TBI 小鼠的 LTBMC 产生的 CFU-GEMM 较少；然而，通过 p16-荧光素酶和β-半乳糖苷酶测量，衰老细胞含量增加的集落的相对百分比增加。因此，20 Gy 胸部照射以及 8 Gy TBI 均可在肺部诱导衰老细胞。对于骨髓，8 Gy TBI 不仅诱导造血细胞衰老，而且诱导集落形成祖细胞衰老。p16+/LUC 小鼠品系提供了一个有价值的系统，可用于比较体内器官之间辐射诱导衰老的动力学，并评估衰老细胞在照射性肺纤维化中的潜在作用。
