红细胞分化相关基因敲除小鼠对低剂量辐射损伤敏感性的研究

Response of EDAG knockout mice to low-dose radiation-induced damage

目的构建红细胞分化相关基因(EDAG)敲除小鼠并研究其对低剂量辐射损伤的敏感性。方法利用锌指核酸酶技术(ZFNs)建立EDAG敲除小鼠模型;通过外周血细胞计数、骨髓细胞的DNA损伤和集落形成能力评价低剂量辐射损伤。对野生型及EDAG敲除小鼠进行剂量率为0.31 Gy/min的X线照射,1 min/d,连续照射7 d,小鼠累计照射剂量为2.17 Gy。在X线照射后第1、3、5、7天称重并进行血常规检测(n=7);照射后第3天分离小鼠骨髓细胞,利用免疫荧光实验检测DNA损伤标志物p-H2A.x的表达水平(n=3);照射后第5天分离小鼠骨髓细胞,接种集落并于7 d后进行集落计数(n=3)。结果成功建立了EDAG敲除小鼠模型并在蛋白水平进行了敲除效果的鉴定;X线照射后第3天,与野生型相比,EDAG敲除小鼠白细胞明显减少,敲除小鼠骨髓细胞DNA损伤标志物p-H2A.x表达水平增加;X线照射7 d后骨髓细胞的集落形成能力降低。结论研究发现EDAG敲除小鼠对低剂量辐射诱导的损伤更加敏感,表现为血细胞数量减少,骨髓细胞成集落能力下降,DNA损伤增加。表明EDAG敲除小鼠作为一种对辐射高度敏感的动物模型,可作为低剂量辐射损伤生物学效应评价的有力工具。

Objective To generate the erythroid differentiation associated gene（ EDAG） knockout mice and analyze their sensitivity to low dose radiation-induced damage. Methods Zinc finger nuclease technology（ ZFNs） was used to produce the EDAG knockout mice. The low dose radiation-induced damage was evaluated by peripheral blood cell counts,DNA damage and colony formation of bone marrow cells. Wild-type and EDAG knockout mice were irradiated with 0. 31 Gy /min X-ray,one minute per day for seven consecutive days,and the cumulative radiation dose was 2. 17 Gy（ n = 7）. The blood cell counts were measured by an automated hemocytometer. DNA damage was detected by immunofluorescence assay with a DNA damage marker p-H2 A. x antibody（ n = 3）. The colony formation ability of bone marrow cells was evaluated with a semi-solid culture medium（ n = 3）. Results A model of EDAG knockout mice was established. Compared to wide type mice,white blood cell counts of EDAG knockout mice decreased significantly while the DNA damage marker p-H2 A. x expression was increased on the third day after X-ray irradiation. The ability of colony-forming was reduced after 7 days of X-ray irradiation. Conclusion Our present study found that EDAG knockout mice are more sensitive to low dose radiationinduced damage as shown by decreased peripheral blood cells counts,reduced colony-forming ability of bone marrow cells,and increased DNA damage. These results suggest that EDAG knockout mice can serve as a powerful tool for evaluation of the biological effects of low-dose radiation damage.