李洋洋 张苡铭 魏孔熙 周婷 何进鹏 丁楠 周谷城 史桐凡 柯宜诚 牛帆 刘永琦 张利英

摘 要 目的：比较黄芪不同有效成分对电离辐射致人骨髓间充质干细胞（BMSCs）DNA损伤的防护作用。方法：采用2 Gy X射线直接辐照BMSCs建立辐射细胞模型。采用CCK-8法检测不同质量浓度（25、50、75、100 μg/mL）黄芪多糖、黄芪皂苷、黄芪黄酮辐射前干预1 d+辐射后干预1～5 d对辐射BMSCs增殖的影响，筛选给药浓度和辐射后继续干预时间。将辐射BMSCs分为辐射组、黄芪多糖组、黄芪皂苷组、黄芪黄酮组，后3组辐射前后均使用适宜的相应药物进行干预，另设空白组进行比较;采用胞浆分裂阻滞微核法检测辐射后干预适宜时间的微核细胞率和细胞微核率，免疫荧光法检测辐射后干预适宜时间细胞中53BP1焦点簇数量，并对不同时间点（0.5、2、12、24 h）的53BP1焦点簇数量进行比较。结果：与空白组比较，辐射组BMSCs的OD值显著降低（P<0.05或P<0.01）;与辐射组比较，50 μg/mL黄芪多糖、黄芪皂苷、黄芪黄酮继续干预2～3 d时BMSCs的OD值均显著升高，其余剂量组仅部分时间点有显著差异（P<0.05或P<0.01）;综合考虑确定给药浓度为50 μg/mL，辐射后继续干预时间为2 d。与空白组比较，辐射组、黄芪多糖组、黄芪皂苷组、黄芪黄酮组微核细胞率和细胞微核率均显著升高，辐射组和黄芪多糖组细胞中53BP1焦点簇数量均显著增加（P<0.01）。与辐射组和黄芪黄酮组比较，黄芪多糖组和黄芪皂苷組微核细胞率、细胞微核率和53BP1焦点簇数量（辐射后干预0.5、2、12 h）均显著降低或减少，且黄芪多糖组微核细胞率和细胞微核率均显著低于黄芪皂苷组（P<0.05）;超过24 h检测不出53BP1焦点簇。结论：黄芪多糖和黄芪皂苷对辐射所致的BMSCs DNA损伤均有防护作用，其中黄芪多糖防护效果优于黄芪皂苷;黄芪黄酮对辐射所致的DNA损伤无防护作用。

关键词 黄芪多糖;黄芪黄酮;黄芪皂苷;电离辐射;骨髓间充质干细胞;DNA损伤

ABSTRACT   OBJECTIVE： To compare the protective effects of different effective components of Astragali radix against DNA damage of human bone marrow mesenchymal stem cells （BMSCs） induced by ionizing radiation. METHODS： 2 Gy X-rays were used to directly irradiate BMSCs to establish a radiation model. CCK-8 method was used to detect the effects of different mass concentrations （25， 50， 75， 100 μg/mL） of astragalus polysaccharide， astragalus saponin and astragalus flavonoids for 1 day before radiation + 1 to 5 days after radiation on the proliferation of BMSCs. The dose concentration and the duration of intervention after radiation were selected. The irradiated BMSCs were divided into radiation group， astragalus polysaccharide group， astragalus saponin group and astragalus flavonoids group. The last three groups were treated with appropriate dosage of corresponding drugs before and 2 days after radiation， and a blank group was set for comparison. Cytoplasmic division arrest micronucleus method was used to detect micronucleus cell rate and cell micronucleus rate after appropriate time of intervention following radiation; immunofluorescence method was used to detect the number of 53BP1 foci in cells after appropriare time of intervention following radiation; the number of 53BP1 foci were compared among different time points （0.5， 2， 12， 24 h）. RESULTS： Compared with blank group， OD values of BMSCs were decreased significantly in radiation group （P<0.05 or P<0.01）. Compared with radiation group， the OD values of BMSCs were significantly increased when 50 μg/mL astragalus polysaccharide， astragalus saponin and astragalus flavonoids continuously intervened radiation for 2-3 days， there was significant difference in other groups at some time point （P<0.05 or P<0.01）. After consideration， drug concentration was determined to be 50 μg/mL， and the continuous intervention time was 2 days after radiation. Compared with blank group， the micronucleus cell rate and cell micronucleus rate of radiation group， astragalus polysaccharide group， astragalus saponin group and astragalus flavonoids group increased significantly， and the number of 53BP1 focus cluster in radiation group and astragalus polysaccharide group increased significantly （P<0.01）. Compared with radiation group and astragalus flavonoids group， the micronucleus cell rate， cell micronucleus rate and the number of 53BP1 focus cluster （continued intervention for 0.5， 2， 12 h） in the astragalus polysaccharide group and astragalus saponin group were significantly reduced， and the micronucleus cell rate and cell micronucleus rate in the astragalus polysaccharide group were significantly lower than astragalus saponin group （P<0.05）. 53BP1 focus cluster could not be detected 24 h later （P<0.05）. CONCLUSIONS： Astragalus polysaccharide and astragalus saponin both have protective effects on BMSCs DNA damage induced by radiation， and the protective effect of astragalus polysaccharide is better than that of astragalus saponin; astragalus flavonoids has no protective effect on radiation-induced DNA damage.

KEYWORDS   Astragalus polysaccharide; Astragalus flavonoids; Astragalus saponin; Ionizing radiation; Bone marrow mesenchymal stem cells; DNA damage

放疗是恶性肿瘤的主要治疗手段之一，放疗在杀伤肿瘤组织的同时，不可避免地会对正常组织造成损伤，产生放疗毒性[1-2]。已有研究表明，电离辐射（Ionizing radiation，IR）可通过多种机制破坏细胞，其中最重要的是造成细胞DNA损伤，当损伤无法修复时，则可引发细胞癌变[3]。骨髓间充质干细胞（Bone marrow mesenchymal stem cells，BMSCs）是具有自我更新和多向分化潜能的细胞，参与多种组织的修复。当辐射肿瘤组织时，该组织周围会聚集大量的BMSCs;受辐射影响，BMSCs可引发多种生物学反应，包括活性氧（ROS）生成、細胞凋亡和增殖抑制等，甚至会发生BMSCs恶性转化[4]。……