| 时磊,申星,董雅,等.经小鼠外周血直接检测淋巴细胞γ-H2AX的方法及应用[J].中华放射医学与防护杂志,2025,45(1):18-23.Shi Lei,Shen Xing,Dong Ya,et al.An experimental method for direct detection of lymphocyte γ-H2AX in mice peripheral blood and its application[J].Chin J Radiol Med Prot,2025,45(1):18-23 |
| 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 lymphocyte γ-H2AX in mice peripheral blood and its application |
| 投稿时间：2024-06-22 |
| DOI：10.3760/cma.j.cn112271-20240622-00232 |
| 中文关键词: 流式细胞术 γ射线 γ-H2AX 剂量效应 |
| 英文关键词:Flow cytometry Gamma rays γ-H2AX Dose-effect relationship |
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| 中文摘要: |
| 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 Gy γ 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(R2=0.991 4)。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。 |
| 英文摘要: |
| Objective To develop a method of employing flow cytometry to directly detect the γ-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, and γ-H2AX was labeled with specific antibodies. DRAQ5 dye was used to further exclude debris and anucleate cells. The mean fluorescence intensity of γ-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 of γ-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 of γ-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 of γ-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 of γ-H2AX at 24 h after irradiation was consistent with the lowest values of the three blood cell lines at 7 or 14 d after irradiation. Conclusions The application of flow cytometry with a fixation/dissolution protocol to directly detect the mean fluorescence intensity of γ-H2AX in peripheral blood lymphocytes of mice has significant application value in radiation biology effect research, radiation protection drug screening, and efficacy evaluation. |
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