氮氧自由基R-1对人肝细胞L-02的辐射防护机制研究

Radioprotective mechanism of nitroxides R-1 on human liver cell L-02

探讨氮氧自由基R-1(下称R-1)对人肝细胞L-02辐射损伤的保护机制,将L-02细胞分成4组:对照组、药物组、照射组和药物+照射组,药物组和照射组分别给予0.25μmol/L的R-1和4Gy的^(60)Coγ射线照射处理,药物+照射组接受这两种处理,对照组不接受处理。流式细胞仪检测各组细胞周期,化学发光法检测超氧化物歧化酶(SOD)的活力、分光光度法检测谷胱甘肽(GSH)的活力、硫代巴比妥酸(TBA)法测定丙二醛(MDA)的含量和DCFH-DA法测量活性氧(ROS)含量;Western blot检测各组P53、Bcl-2、Bax、caspase-3的蛋白表达。结果显示:与照射组相比,药物+照射组的G_2期阻滞缓解(48hG_2期的细胞比例为29.40±3.40:44.01±2.71;t=6.46,p<0.05)、SOD(97.03±5.99:50.94±5.47;t=9.84,p<0.05)和GSH(16.56±0.91:7.49±0.54;t=14.81,p<0.05)的活力增强、MDA(2.87±0.33:4.93±0.17;t=9.56,p<0.05)和ROS(45.33±3.79:75.67±4.70;t=8.67,p<0.05)的含量减少,差异有统计学意义。Bcl-2的表达上调,P53、Bax和caspase-3的表达水平下降。本实验结果表明:氮氧自由基R-1通过清除L-02细胞内的自由基而发挥其辐射防护作用。

In order to explore the radio-protective mechanism of treatment with nitroxides R-1 on the human liver cell L-02 was used as a model of target, and L-02 cells were divided into the control, drug, irradiation and combination groups. The samples of drug and irradiation groups were given 0.25 μmol / L of R-1 and exposed to 4 Gy of 60Co μ-rays irradiation respectively; the combination group received both treatments; whereas there was no any treatment with the control group. The cell cycle was measured by flow cytometry; the enzyme activity of SOD and GSH was detected by chemiluminescence and spectrophotometric method, respectively meanwhile the concentration of MDA and ROS was measured by TBA and DCFH-DA, respectively. The expression of p53, Bcl-2, Bax and caspase-3 protein was observed by western blot. The results show that compared with irradiation group, the G2 cell cycle arrest was relieved （determined at 48 h, G2 phase proportion of the cell was （29.4±3.40）：（44.01±2.71）; t=6.46, p〈0.05, the enzyme activity of SOD was （97.03±5.99）：（50.94±5.47）; t=9.84, p〈0.05 and GSH was （16.56±0.91）：（7.49±0.54）; t=14.81, p〈0.05 meanwhile the concentration of MDA was （2.87±0.33）：（4.93±0.17）; t=9.56, p〈0.05 and ROS was （45.33±3.79）： （75.67±4.7）; t=8.67, p〈0.05. It was found that the expression of p53, Bax and caspase-3 decreased significantly, while the expression of Bcl-2 increased in the combination group. This experiment indicates that nitroxides R-1 can protect the L-02 cell from radiation damage effectively by removing the free radical in cells.