天津医药

天津医药 ›› 2017, Vol. 45 ›› Issue (2): 155-159.doi: 10.11958/20161281

• 实验研究 • 上一篇    下一篇

吲哚-3-甲醇对小鼠骨髓造血细胞辐射损伤的防护作用及其机制

路璐， 董佳丽， 樊赛军△   

  1. 中国医学科学院北京协和医学院放射医学研究所， 天津市放射医学与分子核医学重点实验室 （邮编 300192）
  • 收稿日期:2016-11-04 修回日期:2016-12-20 出版日期:2017-02-15 发布日期:2017-02-14
  • 通讯作者: △通讯作者 E-mail: fansaijun@irm-cams.ac.cn E-mail:0lulu0@163.com
  • 作者简介:路璐 （1982）， 女， 助理研究员， 硕士， 主要从事辐射损伤防护的药物研究
  • 基金资助:
    国 家 自 然 科 学 基 金 面 上 项 目（81071906， 81172127， 81572969）；天 津 市 应 用 基 础 与 前 沿 技 术 研 究 计 划 青 年 项 目（15JCQNJC46000）； “协和青年基金资助” 和 “中央高校基本科研业务费专项资金资助” （3332013044）； 中国医学科学院、 北京协和医学院 “中央级公益性科研院所基本科研业务费” （2016RC310016）

The effect and mechanism of indole-3-carbinol on alleviating the radiation-induced bone marrow hematopoietic cell injury in mice

LU Lu, DONG Jia-li, FAN Sai-jun△   

  1. Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical Collage, Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Tianjin 300192, China
  • Received:2016-11-04 Revised:2016-12-20 Published:2017-02-15 Online:2017-02-14
  • Contact: △Corresponding Author E-mail: fansaijun@irm-cams.ac.cn E-mail:0lulu0@163.com

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摘要： 目的 研究吲哚-3-甲醇 （I3C） 对小鼠骨髓造血细胞辐射损伤的保护作用及机制。方法 （1） 密度梯度离心法获得 CD45.1 亚型 C57BL/6J 小鼠骨髓有核细胞， 经 0 mol/L、 10-8 mol/L ~10-3 mol/L I3C 处理后， 接受不同剂量（0 Gy、 1 Gy、 4 Gy）的 137Cs γ-射线照射； 继续培养 18 h 后采用生物发光法检测细胞活力。（2）设空白对照组和 10-6 mol/L I3C 组， 经上述 3 种剂量射线照射后， 接种于甲基纤维素半固体培养基中培养 7 d， 观察骨髓粒-单核巨噬细胞集落（CFU-GM） 形成情况。（3） 取 24 只 CD45.2 亚型小鼠接受 8 Gy 137Cs γ-射线照射作为受体， CD45.1 亚型小鼠骨髓有核细胞 （供体） 设空白对照组、 4 Gy 照射组和 4 Gy 照射+10-6 mol/L I3C 组。将供体与竞争者 （CD45.2 亚型） 骨髓细胞混和后， 接种于受体小鼠体内 （每组 8 只）， 流式细胞术检测受体小鼠外周血细胞中供体来源的细胞比例。（4） 细胞设空白对照组、 10- 6 mol/L I3C 组、 1 Gy 照射组和 1 Gy 照射+10- 6 mol/L I3C 组。培养 24 h 后收集细胞， 提取蛋白后 Western blot 法检测各组核因子 NF-E2 相关因子 （Nrf2）、 血红素加氧酶 （HO） -1 表达。结果 （1） I3C 浓度>10-4 mol/L 时出现明显的细胞毒性作用 （P > 0.05）； 相同剂量射线照射下， 10-7~10-6 mol/L I3C 可减轻射线对细胞的损伤； 因此选取 10-6 mol/L 为本研究 I3C 的实验浓度。（2） 相同剂量射线照射下， 10-6 mol/L I3C 组 CFU-GM 形成数量较空白对照组明显升高（P < 0.05）。（3）流式细胞结果显示， 4 Gy 照射组细胞移植后， 受体小鼠外周血中供体细胞比例较对照组明显降低（P < 0.05）， 而 10-6 mol/L I3C 预处理的供体小鼠细胞移植后， 受体小鼠中供体细胞比例较 4 Gy 照射组升高（P < 0.05）。（4） Western blot 结果显示， 1 Gy 照射+10-6 mol/L I3C 组 Nrf2 和 HO-1 蛋白表达水平明显高于其他 3 组（P < 0.05）。结论 I3C 可以减轻辐射引起的小鼠造血细胞损伤和功能下降， 其机制可能与激活 Nrf2/HO-1 通路有关。

关键词: 骨髓祖代细胞, 辐射损伤, 辐射防护, 造血干细胞, 造血祖细胞, 吲哚-3-甲醇

Abstract: Objective To investigate the protective effect of indole- 3- carbinol (I3C) on radiation- induced mouse bone marrow hematopoietic cell injury and the involved mechanisms. Methods (1) The bone marrow nuclear cells (BMNCs) from CD45.1 subtype of C57BL/6J mice were collected by a density gradient centrifugation method. The BMNCs were pretreated with a series doses of I3C (0 mol/L, 10- 8 mol/L-10- 3 mol/L) and then exposed with radiation of 137Cs γ-ray (doses of irradiation were 0 Gy, 1 Gy and 4 Gy). After 18-hour culturing, the bioluminescence method was used to detect the cell viability. (2) These cells were divided into control group and 10- 6 mol/L I3C group. Both groups were received the irradiation (0 Gy, 1 Gy and 4 Gy) and inoculated into the methylcellulose semi-solid culture medium to incubate 7 days, the colony forming unit-granulocyte monocytes (CFU-GM) were observed. (3) Twenty-four CD45.2 subtype mice used as the receptor were exposed with 8 Gy radiation. The CD45.1 BMNCs were divided into control group, 4 Gy irradiation group, 4 Gy irradiation and 10- 6 mol/L I3C group. Donor cells were harvested from C57BL/6J (CD45.1) mice after they received various treatments, and were then mixed with competitive BMNCs from C57BL/6J (CD45.2) mice. The mixed cells were transplanted into recipient mice (8 mice/group). Flow cytometry was used to analyze the proportion of donor cells in peripheral blood of receptor. (4) The cells were divided into control group, 10- 6 mol/L I3C group, 1 Gy irradiation group, 1 Gy irradiation with 10-6 mol/L I3C group. After 24-hour culturing, Western blot assay was used to detect the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Results (1) I3C showed a significant cytotoxic effect on the BMNCs when its concentration was above 10- 4 mol/L. 10- 7-10- 6 mol/L I3C could reduce the radiation injury of BMNCs under the same dose of irradiation. Therefore, 10-6 mol/L I3C was chosen for subsequent experiments. (2) The CFU-GM was significantly higher in 10-6 mol/L I3C group than that of control group (P < 0.05). (3) Results of flow cytometry showed that the proportion of donor cells in receptor was significantly higher in 4 Gy irradiation group than that of control group, which decreased the engraftment capability of irradiated HSCs (P < 0.05), although the engraftment capability of irradiated HSCs improved after 10-6 mol/L I3C treatment. (4) I3C significantly enhanced the increased protein expression of Nrf2 and HO-1 caused by radiation (P < 0.05). Conclusion I3C has a protective effect on hematopoietic cells following radiation-induced injury, which may be related with activating the Nrf2/HO-1 signal pathway.

Key words: myeloid progenitor cells, radiation injuries, radiation protection, hematopoietic stem cells, hematopoietic progenitor cells, indole-3-carbinol