氮氧自由基R-1对人肝细胞L-02的辐射防护作用  

Radioprotective effects of nitroxides R-1 on human liver cells L-02

作　　者：侯知佑[1] 李静[1] 刘军叶[1] 周咏春[1] 刘海强[1] 张琰君[1] 孙晓莉[1] 郭国祯[1] 

机构地区：[1]第四军医大学军事预防医学系放射医学教研室,西安710032

出　　处：《中华放射医学与防护杂志》2011年第2期172-176,共5页Chinese Journal of Radiological Medicine and Protection

基　　金：国家自然科学基金（60871086）

摘　　要：目的探讨氮氧自由基R-1（简称R-1）对人肝细胞L-02的辐射防护作用。方法在L-02细胞培养液中，加入终浓度为0、0．125、0．25、0．5、1、2、4、8、16、32μmol／L的R-1，作用24、48和72h。用MTT法测定R-l的毒性作用，以筛选合适的R-1浓度。后续实验选择0．125、0．25、0．5、1Ixmol／L4个浓度组检测其防护作用。设终浓度4mmol／L的细胞保护剂WR2721为阳性对照组。采用”60^Co γ射线照射，吸收剂量为0、1、2、4、8Gy，照射72h后，进行MTT比色法。L-02细胞分为2组：照射前30 min加药组和照射后立即加药组。终浓度均为0.25Ixmol／L，吸收剂量为4Gy，照射后72h进行MTT比色实验。照射后10d，用克隆形成实验检测不同浓度的R-1对L-02细胞活力的影响。选用防护效果最佳的0.25ixmoL／L浓度对细胞进行预处理，分别在4Gy照射后的24、48和72h，用倒置显微镜观察细胞形态的变化，Hoechst33258荧光染色法和流式细胞仪检测细胞凋亡。结果当R-1浓度低于1μmol／L时，与0 μmol／L组相比，L-02细胞各时间点的吸光度（A）值无明显变化；而浓度高于2txmol／L时，与0txmol／L组相比，其A值随浓度的增高而下降。选用0、0．125、0．25、0．5、1Ixmol／L的浓度组，与照射组相比，R-1各浓度组的A值和克隆形成率明显提高，其中0．25pμmol／L组的作用最明显。与照射组相比，0.25Iμmol／L预处理组的L-02细胞贴壁好，折光性强，轮廓清晰，凋亡细胞和死亡细胞明显较少。结论R-1能有效地防护”co1射线对L-02细胞的辐射损伤，其防护作用可能与减少细胞凋亡有关。Objective To investigate the protective effects of the nitroxides R-1 on human liver cells exposed to ionizing radiation. Methods Human liver ceils L-02 were cultured and irradiated with ^60Co γ-rays at the doses of 0, 1,2, 4, and 8 Gy, in order to screen the proper irradiation dose. WR2721 at the terminal concentration of 4 mmoI/L was used as positive control. L-02 cells irradiated with 4 Gy were added with R-1 at the terminal concentration of 0. 25 μmol/L at 30 rain before irradiation or immediately after irradiation. MTT method was used to screen the proper conditions for follow-up experiment 72 h later. L-02 cell culture fluid was added with R-1 at the concentrations of 0, 0. 125, 0. 25, 0. 5, and 1 μmol/L, respectively for 30 rain before irradiation at the doses of 0, 1, 2, 4, and 8 Gy to calculate clone formation rate at 10 d post-irradiation. L-02 cells were cultured and divided into 4 groups： control group without any treatment, drug group pretreated by 0. 25 μmol/L R-1 only, irradiation group, irradiated at 4 Gy only, and drug ＋ irradiation group with combination of 0.25 μmol/L R-01 and 4 Gy irradiation. The inverted microscopy and Hoechst 33258 staining and flow cytometry were used to observe the apoptosis of the cells at 24, 48, and 72 h later. Results Nitroxides R-1 did not inhibit the viability of L-02 cell when its concentration was less than 1 μmol/L and it inhibited the L-02 cell growth when the concentration was higher than 2 μmol/L. The A value＇and colony formation rate of different concentration of R-1 groups were all higher than those of the irradiation group, and the effect of the 0. 25 μmol/L drug concentration groupwas the most significant. Consequently, the concentration 0.25 txmol/L was selected for follow-up experiment. Compared with the irradiation group, the l,-02 cells of the pretreatment group showed solid adherence, increased refraction, clear outline, less apoptotic and dead cells at 4 Gy post-irradiation.

关 键 词：氮氧自由基 人肝细胞 辐射防护 Γ射线 凋亡 

分 类 号：R144[医药卫生—公共卫生与预防医学]