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

作　　者：张晋福 徐子松 范汉程 杨子豪 邓榕 曾俊文 舒鑫 曾慧红[2,3] 邵立健 ZHANG Jinfu;XU Zisong;FAN Hancheng;YANG Zihao;DENG Rong;ZENG Junwen;SHU Xin;ZENG Huihong;SHAO Lijian(Department of Occupational Health and Environmental Health/Jiangxi Provincial Key Laboratory of Preventive Medicine,School of Public Health,Nanchang University,Nanchang,Jiangxi 330006,China;Department of Histology and Embryology,School of Basic Medicine,Nanchang University,Nanchang,Jiangxi 330006,China;Provincial Key Laboratory of Micro-Nano Science and Technology,Institute for Advanced Study,Nanchang University,Nanchang,Jiangxi 330006,China)

机构地区：[1]南昌大学公共卫生学院,劳动卫生与环境卫生学教研室/江西省预防医学重点实验室,江西南昌330006 [2]南昌大学基础医学院组织胚胎学教研室,江西南昌330006 [3]南昌大学高等研究院微纳米科学与技术省重点实验室,江西南昌330006

出　　处：《环境与职业医学》2022年第7期792-798,共7页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金(82073484,81860026);江西省自然科学基金(20192BBG70043,20202ACB206009);南昌大学交叉创新基金项目(9166-27060003-YB13)。

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�[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>

关 键 词：电离辐射 造血干/祖细胞 细胞周期 活性氧 细胞老化 

分 类 号：R13[医药卫生—劳动卫生]