| 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].Chinese Journal of Radiological Medicine and Protection,2025,45(1):18-23 |
| An experimental method for direct detection of lymphocyte γ-H2AX in mice peripheral blood and its application |
| Received:June 22, 2024 |
| DOI：10.3760/cma.j.cn112271-20240622-00232 |
| KeyWords:Flow cytometry Gamma rays γ-H2AX Dose-effect relationship |
| FundProject: |
| Author Name | Affiliation | E-mail | | Shi Lei | 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 | | | Shen Xing | Institute of Radiation Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China | | | Dong Ya | Graduate Training Base of Jinzhou Medical University/PLA Rocket Army Characteristic Medical Center, Beijing 100088, China | | | Zhang Qiaoyun | Clinical Laboratory, PLA Rocket Force Characteristic Medical Center, Beijing 100088, China | | | Ou Hongling | Clinical Laboratory, PLA Rocket Force Characteristic Medical Center, Beijing 100088, China | | | Song Xiujun | Clinical Laboratory, PLA Rocket Force Characteristic Medical Center, Beijing 100088, China | | | Ma Yingying | Clinical Laboratory, PLA Rocket Force Characteristic Medical Center, Beijing 100088, China | | | Wang Xinru | Clinical Laboratory, PLA Rocket Force Characteristic Medical Center, Beijing 100088, China | wangxinru@126.com |
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| Abstract:: |
| 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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