Sirt3介导的SOD2在电离辐射中的作用研究

摘要

目的 观察293T细胞经不同剂量137Cs γ射线照射后,细胞中沉默信息调节因子3(sirt3)及相关抗氧化因子的变化,研究sirt3基因在辐射防护中的抗氧化活性机理.方法 采用137Cs对293T细胞分别进行2、4、8和16 Gy照射,采用Real-Time PCR的方法,检测照后6、12、24和48 h,细胞中sirt3和超氧化物歧化酶2(SOD2) mRNA的表达水平变化;采用免疫印迹法,检测细胞中sirt3和SOD2蛋白水平随时间的变化;采用siRNA干扰和抑制剂处理的方法,检测sirt3与SOD2的作用关系;采用SOD Assay Kit-WST试剂盒检测细胞中SOD2酶活力的变化;流式细胞术检测细胞中活性氧(ROS)随时间的变化.结果 各组细胞中sirt3和SOD2的mRNA水平和蛋白质水平均表现出不同程度的上升,与0 Gy组比较,分别在12 h(t =6.75、13.59、6.59、10.13,P＜0.05)和24 h(t=6.80、8.73、11.09,P＜0.05)达到最大值;sirt3 siRNA干扰和抑制剂处理结果显示sirt3是SOD2的上游调控因子;照射24 h后细胞中SOD2的活性显著增加,与0 Gy组比较,差异有统计学意义(t =46.04、23.19、26.28、14.70,P＜0.05),细胞中ROS水平也发生相应变化.结论 sirt3可能通过对细胞中SOD2的表达量及活性的调控加强抗氧化保护系统,为临床辐射防护和治疗提供实验依据.%Objective To study the effects of the silent information regulator 3 (sirt3) and its relative anti-oxidation factors on ionizing radiation (IR)-induced damage in 293T cells.Methods 293T cells irradiated with 2,4,8,16 Gy of γ-rays,after 6,12,24 and 48 h of exposure,the expressions of sirt3 mRNA and SOD2 protein were examined by Real-Time PCR and immunoblotting assay,respectively.sirt3 siRNA was applied to knock down sirt3 in the cells.SOD2 activities were measured by a WST kit and the cellular ROS level was detected by a flow cytometry.Results The mRNA levels of sirt3 and SOD2 in each group campared with the 0 Gy group were reached the maximum at 12 h (t =6.75,13.59,6.59,10.13,P ＜ 0.05) and 24 h(t =6.80,8.73,11.09,P ＜ 0.05),and protein levels also increased.It was found that sirt3 was a upstream regulatory factor of SOD2.The activity of SOD2 were much higher at 24 h (t=46.04,23.19,26.28,14.70,P ＜ 0.05) than that in the 0 Gy group as well as the level of ROS.Conclusion Sirt3 may strengthen antioxidant protection system through regulation of the SOD2 expression and activity,and possibly plays an important role in IR-induced damage response and clinic treatment.