不同剂量辐射对小鼠造血干/祖细胞的慢性损伤
Chronic injury of hematopoietic stem and progenitor cells induced by different doses of radiation
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摘要:
背景 电离辐射（IR）引起的造血系统慢性损伤常被忽略，该损伤的本质原因是造血干/祖细胞（HSPCs）的损害。
目的 探究不同辐射剂量与同一辐射剂量不同辐射方式IR对小鼠骨髓内HSPCs的长期影响，为减少IR导致的造血系统慢性损伤提供科学依据。
方法 将16只8~10周龄雄性C57BL/6小鼠随机分为4组。各组小鼠接受不同剂量和不同方式的X射线全身辐射，分别为1.5 Gy连续4次（1.5 Gy×4）辐射组（n=5）、3 Gy辐射组（n=4）、6 Gy辐射组（n=4）和未辐射组（n=3）。辐射后2个月，收集各组小鼠骨髓细胞并计数，通过体外克隆形成实验（CAFC）分析骨髓细胞的克隆形成能力，通过流式细胞术分析HSPCs的细胞比例，通过增殖细胞核抗原-67（Ki-67）和7-氨基放线菌素D（7-AAD）双染色分析HSPCs的细胞周期，通过2, 7-二氯二氢荧光素二乙酸酯（DCFDA）探针分析HSPCs的活性氧（ROS）水平，通过5-十二酰氨基荧光素-二-β-D-吡喃半乳糖苷（C12FDG）探针分析HSPCs的细胞老化情况，通过实时荧光定量PCR检测P16、P19、P21和P27等老化相关基因的表达差异。
结果 不同剂量和不同方式辐射小鼠后2个月，小鼠骨髓细胞数无明显变化（均P>0.05）；3 Gy和6 Gy辐射后骨髓细胞的体外克隆形成能力较未辐射组下降（P<0.01）；HSPCs对不同辐射剂量和辐射方式的反应不一致。总体来看，与未辐射组相比，辐射后长时程造血干细胞（LT-HSCs）细胞比例无明显变化（P>0.05），造血祖细胞（HPCs）、造血干细胞（HSCs）、短时程造血干细胞（ST-HSCs）和多能造血祖细胞群2（MPP2）细胞比例升高（均P<0.05），LSK、MPP1、MPP3和MPP4细胞比例降低（均P<0.05）；除了HPCs和MPP2外，HSPCs的G0期比例减少（均P<0.05）；单次6 Gy辐射后HSPCs的ROS明显增加（均P<0.05），3 Gy和1.5 Gy×4辐射后的ROS与未辐射组水平无差异（均P>0.05）；HPCs、LSK和HSCs细胞经辐射暴露后老化细胞比例均增加（均P<0.05），且1.5 Gy×4和6 Gy辐射后HSCs内P16、P19、P21和P27等老化相关基因表达量较未辐射组升高（均P<0.05）。
结论 骨髓内HSPCs对不同剂量和不同方式的IR反应不一致。ROS累积和细胞老化可能参与了IR导致的HSPCs损伤过程。
Abstract:Background The chronic injury of the hematopoietic system caused by ionizing radiation (IR) is often ignored. The essential cause of this injury is the damage of hematopoietic stem and progenitor cells (HSPCs).
Objective To explore the long-term effects of IR at different radiation doses and at different radiation fractions of the same radiation dose on HSPCs in the bone marrow of mice, and to provide a scientific basis for reducing the chronic damage to the hematopoietic system caused by IR.
Methods A total of 16 male C57BL/6 mice aged 8-10 weeks were randomly divided into four groups that received different doses or fractions of total body X-ray irradiation, including 1.5 Gy×4 irradiation group (n=5), 3 Gy irradiation group (n=4), 6 Gy irradiation group (n=4), and non-irradiation group (n=3). Two months after irradiation, bone marrow cells from each mouse were collected and counted. The clone forming ability of bone marrow cells was analyzed by cobblestone area-forming cell (CAFC) assay. The proportion of HSPCs was measured by flow cytometry. The cell cycle of HSPCs was assessed by antigen identified by monoclonal antibody Ki 67 (Ki-67) and 7-amino-actinomycin D (7-AAD) double staining. The reactive oxygen species (ROS) levels of HSPCs were estimated with a 2,7-dichlorodihydrofluorescein diacetate (DCFDA) probe. The cellular senescence of HSPCs was evaluated with a 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG) probe. The expression of senescence related genes such as P16, P19, P21, and P27 was measured by real-time fluorescence quantitative PCR.
Results There was no significant change in the numbers of bone marrow cells 2 months after different doses and fractions of radiation (P>0.05). The clone forming ability of bone marrow cells was significantly decreased after 3 Gy and 6 Gy irradiation when compared to non-irradiated mice (P<0.01). HSPCs responded inconsistently to different doses and fractions of irradiation. Overall, there was no significant change in long-term hematopoietic stem cells (LT-HSCs) proportion after irradiation (P>0.05), the proportions of hematopoietic progenitor cells (HPCs), hematopoietic stem cells (HSCs), short-term hematopoietic stem cells (ST-HSCs), and multipotent progenitors 2 (MPP2) increased after irradiation (P<0.05), and the proportions of LSK, MPP1, MPP3, and MPP4 cells decreased after irradiation (P<0.05); except for HPCs and MPP2, the proportion of HSPCs in G0 phase was decreased (P<0.05). The ROS production in HSPCs was increased significantly after 6 Gy irradiation (P<0.05), while the ROS levels after 3 Gy and 1.5 Gy×4 irradiation were similar to that of the non-radiation group (P>0.05). The cellular senescent proportion of HPCs, LSK, and HSCs increased after irradiation (P<0.05). The expression levels of senescence related genesP16, P19, and P21 in HSCs were significantly increased (P<0.05).
Conclusion The responses of HSPCs in bone marrow to IR vary depending on doses and fractions of irradiation. Increased ROS production and cellular senescence may be involved in the damage process of HSPCs under radiation settings.
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