花青素对电离辐射引起骨髓c-kit阳性细胞损伤防护作用的体外研究

摘要

Objective To observe the protective effect of anthocyanin on irradiation induced bone marrow c-kit positive cell injury, and further explore its possible mechanism. Methods Mouse bone marrow c-kit positive cells were collected by cell sorting method. There were 2 groups: control group and anthocyanin group, which were sub-divided into three groups and received 0 Gy, 1 Gy and 4 Gy irradiation respectively. The control group was added 700μL cell suspension and an equal volume of serum-free hematopoietic stem/progenitor cell culture medium. The 2 × 10-5 mol/L anthocyanin was co-cultured with mouse bone marrow c-kit positive cells of anthocyanin group half an hour before irradiation exposure, then cells were cultured for 18 hours under the conventional culture conditions (37℃,5%CO2). Mouse c-kit positive cell viability was measured by bioluminescence, and which was reflected by relative light units (RLU). The ability of colony-forming units was reflected by CFU-GM. The reactive oxygen species (ROS) level and mean fluorescence intensity (MFI) ofγ-H2AX were detected by flow cytometry. Results Compared to un-irradiated control group, the cell viability and the number of CFU-GM were decreased significantly, while the ROS level and MFI ofγ-H2AX were increased in c-kit positive cells irradiated with 1 Gy and 4 Gy (P<0.05). Compared to 1 Gy and 4 Gy irradiation groups, c-kit positive cell viability and the number of CFU-GM were increased, the ROS level and MFI of γ-H2AX were decreased in anthocyanin group (P < 0.05). Conclusion Anthocyanin exhibits a promising protective effect on radiation-induced bone marrow c-kit positive cell injury, which may be related to the alleviating ROS and DNA damage in bone marrow cells.%目的：观察花青素对电离辐射致小鼠骨髓c-kit阳性细胞损伤的防护作用及可能的作用机制。方法经磁珠细胞分选法获得小鼠骨髓c-kit阳性细胞，分为对照组和花青素组，每组再分成3份，分别接受0、1及4 Gy的137Csγ射线照射。对照组加700μL细胞悬液及等体积的无血清造血干/祖细胞扩增培养基，花青素组加700μL细胞悬液及含2×10-5 mol/L花青素的等体积无血清造血干/祖细胞扩增培养基（37℃，5%CO2），照射前30 min加入，照射后继续培养18 h。采用化学发光法检测小鼠骨髓c-kit阳性细胞的细胞活力，以相对荧光强度（RLU）表示。甲基纤维素半固体培养法检测克隆形成数目（CFU-GM）。流式细胞术检测细胞内活性氧（ROS）及磷酸化组蛋白H2AX（γ-H2AX）平均荧光强度。结果组内比较：与0 Gy比较，经1 Gy和4 Gy照射后小鼠骨髓c-kit阳性细胞活力下降，CFU-GM下降，细胞内ROS增加，γ-H2AX平均荧光强度增加（P＜0.05）。组间比较：与1 Gy和4 Gy对照组比较，花青素1 Gy和4 Gy组骨髓c-kit阳性细胞活力增加，CFU-GM增加，细胞内ROS降低，DNA损伤程度（γ-H2AX平均荧光强度）降低（P＜0.05）。结论花青素通过降低细胞内ROS水平及DNA损伤程度起到对受照射小鼠骨髓c-kit阳性细胞的防护作用。