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造血系统包括造血细胞、造血微环境(又称为造血干细胞龛)和造血刺激因子,其对电离辐射非常敏感。电离辐射主要破坏或抑制造血细胞的增殖能力,而骨髓抑制是造血系统辐射损伤的主要表现。造血干细胞(hematopoietic stem cell,HSC)具有自我更新、增殖和分化生成全血细胞的功能,其受损伤后将引起骨髓持久性抑制,甚至造成个体死亡[1]。大量研究结果表明,HSC储存在造血干细胞龛中[2]。哺乳动物HSC在骨髓乏氧区骨内膜龛中保持静止状态[3]。正常HSC能够保持细胞内低氧浓度并稳定表达低氧诱导因子(hypoxia-inducible factor,HIF)。在缺少HIF-1α的小鼠中,HSC退出细胞循环的静止期,在骨髓移植、骨髓抑制等情况下HSC数量明显降低[4-5]。提高HIF-1α的表达水平,可以提高HSC的归巢和移植再生能力[6-7]。
HIF是一种在低氧浓度下被激活的转录因子,由α亚基和β亚基构成。HIF-1α的转录活性受环境氧浓度的调节。低氧诱导因子抑制因子(factor inhibiting hypoxia-inducible factor,FIH)又名天冬酰胺酰羟化酶,是体内调节HIF-1α转录活性的主要因子。在正常氧浓度下,FIH通过使HIF-1α的C末端反式激活结构域内第803位的天冬氨酸残基羟基化,阻止HIF-1α与转录辅助激活因子CBP/P300结合,从而抑制HIF-1α的转录激活;在低氧浓度下,FIH的活性降低,激活HIF-1α的转录活性,启动低氧应激调控网络[4-6]。研究结果表明,提高HIF-1α蛋白的稳定性,可以缓解辐射引起的造血系统损伤和胃肠功能障碍,减轻放射性胃肠损伤[5, 8-9]。
N-草酰基-D-苯丙氨酸(N-oxalyl-D-phenylalanine,NOFD)是FIH的特异性抑制剂[10],能提高HIF-1α的转录活性。我们的前期研究结果表明,NOFD能够降低辐射后人结肠癌细胞(HCT116)和仓鼠卵巢细胞(CHO-K1)的细胞内活性氧(reactive oxygen species,ROS)水平,减轻辐射引起的DNA损伤和细胞凋亡;NOFD能够促进HCT116细胞中HIF-1α调控的下游基因的表达,包括促红细胞生成素(EPO)、血红素氧合酶1(HO-1)、葡萄糖转运蛋白1(Glut-1)、血管内皮生长因子(VEGF)、Notch2和Notch1;体内实验结果表明,以5 mg/kg的剂量腹腔给予NOFD,可以明显提高辐射损伤小鼠的存活率[11]。本研究旨在系统评价NOFD对小鼠造血系统辐射损伤的防护作用,以期为FIH抑制剂作为新型辐射防护药的研发提供实验依据。
N-草酰基-D-苯丙氨酸对小鼠造血系统辐射损伤的防护作用
Protective effects of N-oxalyl-D-phenylalanine on hematopoietic system injury induced by ionizing radiation in mice
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摘要:
目的 研究N-草酰基-D-苯丙氨酸(NOFD)对小鼠造血系统辐射损伤的防护作用。 方法 将18只6 ~ 8周龄的健康C57BL/6J雄性小鼠按区组随机法分为3组:对照组、4 Gy γ射线全身照射组(简称照射组)和4 Gy γ射线全身照射 + 5 mg/kg NOFD组(简称照射给药组),每组6只。照射给药组于照射前2、16 h及照射后3 d分别腹腔给予5 mg/kg NOFD,对照组和照射组给予等量生理盐水,给药时间和次数与照射给药组相同。采用血细胞计数仪分析各组小鼠外周血血细胞数量;采用流式细胞仪检测外周血中B细胞、T细胞、髓系细胞的百分比,骨髓细胞中造血干细胞(HSC)和造血祖细胞(HPC)的数量及百分比,骨髓细胞中磷酸化组蛋白H2AX(γ-H2AX)和线粒体活性氧(ROS)水平;采用粒细胞-巨噬细胞集落形成单位(CFU-GM)实验和脾集落形成单位(CFU-S)实验检测骨髓细胞的增殖能力。组间两两比较采用Student t 检验。 结果 与照射组相比,照射给药组小鼠外周血中红细胞数量明显增加[(9.05±0.16)×109个/mL对(9.57±0.15)×109个/mL],T细胞的百分比升高[(11.54±0.20)%对(15.31±1.88)%],髓系细胞的百分比降低[(32.67±2.87)%对(24.90±2.19)%],HSC的数量增加[(2.24±0.54)×103个/股骨对(6.77±1.67)×103个/股骨],同时HSC和HPC在骨髓细胞中的百分比[(0.09±0.02)%对(0.59±0.13)%;(0.62±0.14)%对(1.82±0.43)%]升高,且差异均有统计学意义(t=1.998~3.633,均P<0.05)。流式细胞仪检测结果显示,与照射组相比,照射给药组小鼠的骨髓有核细胞(BMNC)和HPC中线粒体ROS自由基(MitoSOX)的平均荧光强度(MFI)明显降低[(6.66±0.56)×103对(3.19±0.25)×103;(2.51±0.46)×103对(1.20±0.35)×103],且差异均有统计学意义(t=6.350、2.282,均P<0.05);同时照射给药组小鼠BMNC、HPC和HSC中γ-H2AX的MFI明显降低[(10.25±0.77)×103对(7.22±0.15)×103;(18.37±2.52)×103对(12.44±0.34)×103;(26.05±2.64)×103对(17.16±1.20)×103],且差异均有统计学意义(t=4.356、2.577、3.070,均P<0.05)。集落形成单位实验结果显示,与照射组相比,照射给药组CFU-GM(12.33±1.48对24.00±3.92)和CFU-S(6.00±1.07对10.83±1.01)明显增加,且差异均有统计学意义(t=2.788、3.288,均P<0.05)。 结论 NOFD对小鼠造血系统辐射损伤有明显的保护作用。 -
关键词:
- 辐射损伤 /
- 造血系统 /
- 活性氧 /
- 辐射防护剂 /
- N-草酰基-D-苯丙氨酸
Abstract:Objective To study the protective effects of N-oxalyl-D-phenylalanine (NOFD) on the radiation injury of hematopoietic system in mice. Methods Eighteen healthy C57BL/6J male mice aged 6−8 weeks were divided into three groups with six mice each according to randomized block design: a control group, a 4 Gy γ ray whole body irradiation group (TBI group), and a 4 Gy γ ray whole body irradiation + 5 mg/kg NOFD group (TBI+NOFD group). The mice in the TBI+NOFD group were intraperitoneally given with 5 mg/kg NOFD at 2, 16 h before irradiation, and 3 d after irradiation, respectively, while the control group and TBI group were intraperitoneally given with the same amount of normal saline at the same time as that in the TBI+NOFD group. The number of peripheral blood cells in each group of mice were analyzed using a blood cell counter. The percentage of B cells, T cells, and myeloid cells in peripheral blood were detected via flow cytometry. The number and percentage of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) in bone marrow cells were detected via flow cytometry. Levels of phosphorylated histone H2AX (γ-H2AX) and mitochondrial reactive oxygen species in bone marrow cells were detected via flow cytometry. The proliferation ability of bone marrow cells was evaluated by counting colony-forming units-granulocyte-macrophage (CFU-GM) and colony-forming units-spleen(CFU-S). Student's t test was used for comparison between two groups. Results Compared with that in the TBI group, the number of peripheral blood erythrocytes in the TBI+NOFD group substantially increased ((9.05±0.16)×109/mL vs. (9.57±0.15)×109/mL). The percentage of T cells increased ((11.54±0.20)% vs. (15.31±1.88)%), whereas the percentage of myeloid cells decreased ((32.67±2.87)% vs. (24.90±2.19)%). The number of HSC increased ((2.24±0.54)×103/femur vs. (6.77±1.67)×103/femur), whereas the percentage of HSC and HPC in bone marrow cells significantly increased ((0.09±0.02)% vs. (0.59±0.13)%, (0.62±0.14)% vs. (1.82±0.43)%; t=1.998−3.633, all P<0.05). The median fluorescence intensity (MFI) of mitochondrial reactive oxygen species (MitoSOX) in bone marrow nucleated cells (BMNC) and HPC in the TBI+NOFD group was significantly lower than that in the TBI group ((6.66±0.56)×103 vs. (3.19±0.25)×103, (2.51±0.46)×103 vs. (1.20±0.35)×103; t=6.350, 2.282, both P<0.05). Moreover, the MFI of γ-H2AX in BMNC, HPC, and HSC in the TBI+NOFD group was significantly lower than that in the TBI group ((10.25±0.77)×103 vs. (7.22±0.15)×103, (18.37±2.52)×103 vs. (12.44±0.34)×103, (26.05±2.64)×103 vs. (17.16±1.20)×103; t=4.356, 2.577, 3.070, all P<0.05). Compared with those in the TBI group, CFU-GM (12.33±1.48 vs. 24.00±3.92) and CFU-S (6.00±1.07 vs. 10.83±1.01) in the TBI+NOFD group significantly increased (t=2.788, 3.288; both P<0.05). Conclusion NOFD exerts an obvious protective effect on the radiation injury of hematopoietic system in mice. -
图 2 N-草酰基-D-苯丙氨酸对辐射损伤小鼠骨髓细胞的影响 a表示与对照组比较,差异均有统计学意义(t=4.503~8.770,均P<0.05);b表示与照射组比较,差异均有统计学意义(t= 2.683、2.581、3.633,均P<0.05)。对照组为未照射且不给药;照射组为4 Gy γ射线全身照射;照射给药组为4 Gy γ射线全身照射+ 5 mg/kg N-草酰基-D-苯丙氨酸。BMNC为骨髓有核细胞;HSC为造血干细胞;HPC为造血祖细胞
Figure 2. Effects of N-oxalyl-D-phenylalanine on bone marrow cells in irradiated mice
图 3 N-草酰基-D-苯丙氨酸对辐射损伤小鼠骨髓细胞中线粒体活性氧水平的影响 a表示与对照组比较,差异均有统计学意义(t=2.538、2.331,均 P<0.05);b表示与照射组比较,差异均有统计学意义(t=6.350、2.282,均 P<0.05)。对照组为未照射且不给药;照射组为4 Gy γ射线全身照射;照射给药组为4 Gy γ射线全身照射+ 5 mg/kg N-草酰基-D-苯丙氨酸。BMNC为骨髓有核细胞;MitoSOX为一种荧光染料;MFI为平均荧光强度;HPC为造血祖细胞;HSC为造血干细胞
Figure 3. Effects of N-oxalyl-D-phenylalanine on the level of mitochondrial reactive oxygen species in bone marrow cells of irradiated mice
图 4 N-草酰基-D-苯丙氨酸对辐射损伤小鼠骨髓细胞DNA损伤的影响 a表示与对照组比较,差异均有统计学意义(t=5.964、4.029、3.465,均 P<0.05);b表示与照射组比较,差异均有统计学意义(t=4.356、2.577、3.070,均 P<0.05)。对照组为未照射且不给药;照射组为4 Gy γ射线全身照射;照射给药组为4 Gy γ射线全身照射+ 5 mg/kg N-草酰基-D-苯丙氨酸。BMNC为骨髓有核细胞;γ-H2AX为磷酸化组蛋白H2AX;MFI:平均荧光强度;HPC为造血祖细胞;HSC为造血干细胞
Figure 4. Effects of N-oxalyl-D-phenylalanine on DNA damage of bone marrow cells in irradiated mice
图 5 N-草酰基-D-苯丙氨酸对辐射损伤小鼠骨髓细胞的CFU-GM和CFU-S的影响 a表示与对照组比较,差异均有统计学意义(t=4.193、2.350,均 P<0.05);b表示与照射组比较,差异均有统计学意义(t=2.788、3.288,均 P<0.05)。对照组为未照射且不给药;照射组为4 Gy γ射线全身照射;照射给药组为4 Gy γ射线全身照射+ 5 mg/kg N-草酰基-D-苯丙氨酸。CFU-GM为粒细胞-巨噬细胞集落形成单位;CFU-S为脾集落形成单位
Figure 5. Effects of N-oxalyl-D-phenylalanine on colony-forming units-granulocyte-macrophage and colony-forming units-spleen in irradiated mice
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