经小鼠外周血直接检测淋巴细胞γ⁃H2AX的方法及应用  

An experimental method for direct detection of lymphocyteγ⁃H2AX in mice peripheral blood and its application

作　　者：时磊 申星 董雅 张巧云 欧红玲 宋秀军 马盈盈 王欣茹 Shi Lei;Shen Xing;Dong Ya;Zhang Qiaoyun;Ou Hongling;Song Xiujun;Ma Yingying;Wang Xinru(Graduate Training Base of Jinzhou Medical University/PLA Rocket Army Characteristic Medical Center,Beijing 100088,China;Clinical Laboratory,PLA Rocket Force Characteristic Medical Center,Beijing 100088,China;Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]锦州医科大学火箭军特色医学中心研究生培养基地,北京100088 [2]火箭军特色医学中心检验科,北京100088 [3]军事科学院军事医学研究院辐射医学研究所,北京100850

出　　处：《中华放射医学与防护杂志》2025年第1期18-23,共6页Chinese Journal of Radiological Medicine and Protection

摘　　要：目的评价流式细胞术经固定/溶解方案检测小鼠外周血淋巴细胞γ⁃H2AX表达水平的方法用于辐射生物学效应研究和辐射防护药物药效评价的可行性。方法C57BL/6J雄性小鼠41只,其中21只按随机数字表法,根据不同照射剂量分为0、1、2、4、6、8、10 Gy组,共7组,每组3只。分别在照射后1、4、8和24 h,对不同剂量γ射线照射小鼠进行尾静脉取血并立即使用甲醛固定,采用Triton X⁃100溶解红细胞,利用γ⁃H2AX特异性抗体进行免疫荧光标记后,使用DRAQ5染料进一步排除碎片和无核细胞,应用流式细胞仪通过前向散射和侧向散射直接分析淋巴细胞群体γ⁃H2AX的平均荧光强度,建立照射后剂量⁃效应曲线。在4和6 Gyγ射线照射条件下,又将另外20只小鼠分为单纯照射组和氨磷汀(WR⁃2721)联合给药组,共4组,每组5只,分别在照后1、4、8和24 h,小鼠进行尾静脉取血,检测淋巴细胞中γ⁃H2AX平均荧光强度,用于评价小鼠的DNA损伤程度和WR⁃2721的防治疗效。结果随着照射剂量增大,小鼠外周血淋巴细胞γ⁃H2AX显著增加,1~2 h后到达峰值,随后降低,量效关系显著(R^(2)=0.9914)。4和6 Gy照后24 h,WR⁃2721给药组γ⁃H2AX平均荧光强度(144.8±8.0)和(109.5±9.7)均低于照射组的(178.0±18.5)和(136.6±5.4),差异有统计学意义为(t=3.78、5.48,P<0.05)。每组小鼠照射后24 h的γ⁃H2AX平均荧光强度与小鼠血常规三系在照后7或14 d的最低值趋势一致。结论应用流式细胞术经固定/溶解方案直接检测小鼠外周血淋巴细胞γ⁃H2AX平均荧光强度,在用于辐射生物学效应研究、辐射防护药物筛选和疗效评价研究中均具有较大的应用价值。Objective To develop a method of employing flow cytometry to directly detect theγ-H2AX expression levels in peripheral blood lymphocytes of mice through fixation and lysis and to evaluate the feasibility of applying this method to research on the radiation-related biological effects and the efficacy evaluation of radioprotective drugs.Methods A total of 41 male C57BL/6J mice were used.First,21 mice were randomly divided into 7 groups according to different radiation doses(0,1,2,4,6,8,and 10 Gy)with 3 mice in each group.Blood samples were collected from the tail vein of mice at 1,4,8,and 24 h after irradiation and immediately fixed with formaldehyde.Red blood cells(RBC)were lysed with Triton X-100,andγ-H2AX was labeled with specific antibodies.DRAQ5 dye was used to further exclude debris and anucleate cells.The mean fluorescence intensity ofγ-H2AX in lymphocyte populations was directly analyzed by flow cytometry through forward and side scatter,and dose-effect curves after irradiation were established.Then,the other 20 mice were divided into radiation alone groups and radiation combined with WR-2721 administration groups at 4 and 6 Gy,respectively,with 5 mice in each group.Blood samples were collected from the tail vein of mice at 1,4,8,and 24 h after irradiation to detect the average fluorescence intensity ofγ-H2AX in lymphocytes,which was used to evaluate the degree of DNA damage in mice and the therapeutic effect of WR-2721.Results The expression ofγ-H2AX in peripheral blood lymphocytes of mice significantly increased with the increase of radiation doses,and reached a peak at 1-2 h and then decreased.The dose-effect relationship was significant(R2=0.9914).At 24 h after 4 and 6 Gy irradiation,compared with the radiation alone groups,the average fluorescence intensity ofγ-H2AX in the radiation combined with WR-2721 administration groups was lower(144.8±8.0 and 109.5±9.7,vs.178.0±18.5 and 136.6±5.4),with statistically significant difference(t=3.78,5.48,P<0.05).The average fluorescence intensity ofγ-H2AX

关 键 词：流式细胞术 Γ射线 γ⁃H2AX 剂量效应 

分 类 号：R144.1[医药卫生—公共卫生与预防医学]