Human lymphocyte damage and phosphorylation of H2AX and ATM induced by γ-rays

Objective To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM.Methods Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy.The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively.The micronucleus(MN)was analyzed by CB method to evaluate DNA damage.Results FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses,and the fitting curve was shown as Y=3.96+11.29D-0.45D2.The level of phosphorylated ATM(p-ATM)was not changed significantly by using the same method.Western blot showed that p-ATM protein expression was significandy increased after irradiation compared with sham.irradiated group.The MN assay which represented DNA damage was sensitive to different doses.Conclusions γ-ray irradiation could induce the phosphorylation of H2AX and ATM,which may play an important role in indicating DNA damage.Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage.     

香兰素衍生物VND3207对不同LET辐射致质粒DNA损伤的防护作用

目的 评价不同LET辐射致DNA损伤以及药物的防护作用.方法 分别以60Co γ射线、质子束、7Li重离子束照射溶液状态超螺旋构象质粒DNA,通过琼脂糖凝胶电泳分析DNA分子构象变化,检测DNA损伤程度及VND3207的防护作用.结果 质子和7Li重离子体外所致质粒DNA损伤明显比γ射线严重.药物VND3207能够有效减轻所观察的3种不同LET辐射对质粒DNA的损伤,加药组与不加药组相比,质粒DNA开环构象显著减少,保护效果随着药物浓度的增加更加明显.200 μmol/L浓度对50 Gy γ射线、质粒和7Li重离子辐照质粒DNA损伤的保护效果(质粒超螺旋构象百分比)分别为85.3%(t=3.70,P=0.033)、73.3%(t=10.58,P=0.017)和80.4%(t=8.57,P=0.008).可见VND3207对重离子辐射损伤的保护效应尤为显著,明显优于质子辐照损伤.结论 药物VND3207对辐射DNA损伤具有较好的保护作用,特别是对γ射线和重离子辐射损伤的保护作用更为明显.

Abstract：

Objective To evaluate the radioprotective effect of vanillin derivative VND3207 on DNA damage induced by different LET ionizing radiation.Methods The plasmid DNA in liquid was irradiated by 60Co γ-rays, proton or 7Li heavy ion with or without VND3207.The conformation changes of plasmid DNA were assessed by agarose gel electrophoresis and the quantification was done using gel imaging system.Results The DNA damage induced by proton and 7Li heavy ion was much more serious as compared with that by 60Co γ-rays, and the vanillin derivative VND3207 could efficiently decrease the DNA damage induced by all three types of irradiation sources, which was expressed as a significantly reduced ratio of open circular form (OC) of plasmid DNA.The radioprotective effect of VND3207 increased with the increasing of drug concentration.The protective efficiencies of 200 μmol/L VND3207 were 85.3% (t =3.70,P =0.033), 73.3% (t = 10.58, P =0.017)and 80.4% (t =8.57,P =0.008)on DNA damage induction by 50 Gy of γ-rays, proton and 7Li heavy ion, respectively.It seemed that the radioprotection of VND3207 was more effective on DNA damage induced by high LET heavy ion than that by proton.Conclusions VND3207 has a protective effect against the genotoxicity of different LET ionizing radiation, especially for γ-rays and 7 Li heavy ion.     

氮氧自由基R-1对人肝细胞L-02的辐射防护作用

Objcetive To investigate the protective effects of the nitroxides R-1 on human liver cells exposed to ionizing radiation.Methods Human liver cells 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 mmol/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 min before irradiation or immediately after irradiation.MIT 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 min before irradiation at the doses of 0,1,2,4,and 8 Gy to ealculate 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 eytometry 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 wu 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 group was the most significant.Consequently,the concentration 0.25 μmoL/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.Conclusions Nitroxides R-1 can protect the human liver cells from 60Coγ-ray induced injury effectively.The mechanism of its protective effect may be the reduction of apoptosis.     

成骨细胞促进急性放射损伤小鼠骨髓造血系统及血管恢复的研究

目的 探讨成骨细胞对放射损伤小鼠骨髓造血系统及血管恢复的影响.方法 取18只雄性BALB/c小鼠股骨制备成骨细胞,其余42只小鼠随机分成健康对照组、成骨细胞组和生理盐水组3组.健康对照组不做任何处理;成骨细胞组和生理盐水组小鼠于6.0 Gy 60 Co γ射线一次性全身均匀照射后,分别经尾静脉输入成骨细胞(2×106个/只)和等体积的生理盐水.于照射后第7、14和21天计数小鼠外周血细胞,骨髓单个核细胞并作骨髓组织学观察.采用流式细胞仪检测骨髓单个核细胞中CD34+细胞百分比,采用免疫组织化学方法检测小鼠骨髓微血管密度.结果 照射后第7、14和21天成骨细胞组小鼠外周血细胞和骨髓单个核细胞计数,骨髓单个核细胞中CD34+细胞百分比,骨髓组织造血面积及骨髓微血管密度均明显高于生理盐水组(t=2.46～64.51,P＜0.05).结论 成骨细胞能促进放射损伤小鼠骨髓造血系统及血管的恢复.

Abstract：

Objective To explore the effects of osteoblasts on the recovery of hematopoiesis and angiogenesis in acute irradiation injury mice.Methods The femurs of 18 male BALB/c mice were used to prepare the bone marrow osteoblasts, and the rest mice were divided into 3 groups as normal group, saline group and osteoblast group.The mice in normal group received no treatment, and the other two groups were received 6.0 Gy 60Co γ-ray irradiation.After irradiation each mouse of osteoblast group was administered with 2 × 106 osteoblasts through tail vein injection, and equal volume saline was given to each mouse of saline group by the same way.The following factors were measured at 7, 14, 21 d after irradiation, they were the counts of peripheral blood cells and bone marrow mononuclear cells ( BMMNC ) , the percentage of CD34 + cells in BMMNC, the histology changes and micro vascular density (MVD) of bone marrow tissue.Results The counts of peripheral blood cells, BMMNC and hematopoietic tissue area in osteoblast group were higher than those in saline group.The percentage of CD34 + cells in BMMNC and the MVD of bone marrow in osteoblast group were also higher than those in saline group at 7, 14, 21 d after irradiation ( t = 2.46 - 64.51, P ＜ 0.05 ).Conclusions Osteoblasts could significantly promote the recovery of hematopoiesis and angiogenesis in mice after acute irradiation injury.     

重组人白细胞介素-11和姜黄素对中子照射致小鼠肠道损伤的治疗作用

Objective To explore the therapeutic effect of recombinant human interleukin(rhIL-11) and curcumin on jejunal damage in mice after neutron irradiation.Methods 140 male BALB/c mice were randomly divided into 4 groups:20 mice in healthy control group,60 mice in mere irradiation group,30 mice in IL-11 treatment group and 30 mice in curcumin treatment group.The mere irradiation group mice were wholly exposed to 3 Gy neutron irradiation.The treatment groups mice were intraperitoneally enterocoelia once a day for 5 d after irradiation.The mortality of the mice were observed.The mice in the control and mere irradiation groups were killed 6 h,1,3,and 6 d post-irradiation,respectively,and the mice of the 2 treatment groups were killed 3 and 6 d post-irradiation,respectively and the samples of jujunum were colleted.HE staining,argyrophilic of nucleaolar organizer regions staining,Feulgen staining,and image analysis were used to observe the pathology and levels of argyrophilic proteins and DNA.Results The mice in the mere irradiation group all died at 5 d post-irradiation,while 2 mice in the IL-11 treatment group and 3 in the curcumin group survived.Large area necrosis and exfoliation were found in the intestinal epithelial mucosa of the mere irradiated group mice since 6 h to 3 d after irradiation.Crypt cell regeneration was seen occasionally found 3 days later and much more 5 days later.Crypt cell regeneration was obviously found in the intestinal epithelial mucosa and lots of new villi were observed 5 d after irradiation in both treatment groups,however,the amounts of crypt cells and new villi of the curcumin treatment group were less than those of the IL-11 treatment group.The contents of AgNOR and DNA in the intestinal epithelial cells 5 days after irradiation of the 2 treatment groups were all significantly higher than those of the mere irradiation group (F = 0.015-0.035,all P ＜ 0.05) but without significant differences between them.Conclusions Jejunal damage in mice could be induced after 3 Gy neutron irradiation.rhIL-11 and curcumin might reduce the damage and promote the regeneration and repair of the intestinal epithelium.     

γ射线诱导人淋巴细胞损伤及磷酸化组蛋白H2AX和ATM表达

目的 探讨人外周血经不同剂量60co γ射线照射后淋巴细胞损伤情况,以及与磷酸化的H2AX、ATM表达的关系.方法 永生化淋巴细胞及人新鲜外周血淋巴细胞经0～8 Gy 60 Coγ射线照射后0.5 h,分别采用Western blot和流式细胞术检测磷酸化ATM和γ-H2AX的表达情况,并通过CB微核法检测受照射后人外周血淋巴细胞微核验证细胞损伤程度.结果 流式细胞检测发现人外周血淋巴细胞照射后0.5 h,γ-H2AX表达的剂量效应关系呈线性平方模式,其拟合曲线为:Y=3.96+11.29D-0.45D2,而相同方法检测磷酸化ATM蛋白的表达未见明显变化.Western blot检测结果表明,照射后0.5 h,各受照射组磷酸化ATM表达在0.1～8 Gy范围内具有呈剂量依赖性增高的趋势.人外周血淋巴细胞微核结果显示,γ射线照射后DNA损伤情况明显加重,随吸收剂量的增加,微核细胞率明显增多.结论 60Co γ射线可诱导DNA双链断裂,随着吸收剂量的增加,微核率明显增加,磷酸化的H2AX、ATM表达水平亦明显增高,本研究为辐射损伤及辐射事故生物剂量估算领域的研究提供了理论依据.

Abstract：

Objective To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM.Methods Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy.The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively.The micronucleus(MN)was analyzed by CB method to evaluate DNA damage.Results FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses,and the fitting curve was shown as Y=3.96+11.29D-0.45D2.The level of phosphorylated ATM(p-ATM)was not changed significantly by using the same method.Western blot showed that p-ATM protein expression was significandy increased after irradiation compared with sham.irradiated group.The MN assay which represented DNA damage was sensitive to different doses.Conclusions γ-ray irradiation could induce the phosphorylation of H2AX and ATM,which may play an important role in indicating DNA damage.Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage.     

电离辐射致股骨头坏死早期病理特点和机制的研究

Objective To observe the earlier pathological character and mechanism of radiation osteonecrosis in femoral head, in order to provide evidences for the earlier diagnosis and prevention of radiation osteonecrosis of femoral head. Methods Single femoral head of rats were irradiated singly with 30 Gy of 137 Cs γ-ray. The rats were executed after 2, 6 and 12 weeks, then the femurs were stained with HE and histopatholngical changes were observed by light microscope. The bone marrow mesenchymal stem cells (BMSCs) were cultured after 2 weeks and its proliferation and the colony formation were observed. The rats were endo-perfused with microfili contrast medium 12 weeks later, and the 3-dimensional structure of capillaries by Micro-CT was re-estabhshed to detect the pathological changes of capillaries after irradiation. Result The irradiated femur showed deranged cbondrocyte, decreased osteocyte, shrinking nucleus, increased empty bone lacuna and reduced bone trabocnla (P < 0.05). Micro-CT showed the discontinued small vessels and absence(6.65 %) capillaries in irradiated femur were obviously less than those of the unirradiated (12.3 %)(P < 0.001). The proliferation of BMSCs was slowed, the number of colony in irradiated group (10 %) was less than that of control (21 %) (P < 0.001). Conclusions The preliminary histopathological changes of osteoradionecrosis on femoral head could be increased the empty bone lacuna, and the bone lacuna above 30 % was the sign of the earlier period of osteoradionecrosis. The osteonecrosis of femoral head induced by radiation is not only correlated to the damages to the bone, but also to the damages to BMSCs and capillaries.     

Therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objectivc To observe the therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy60 Co γ-rays irradiation in beagles,and to provide experimental evidences for the clinical treatment of extremely severe myeloid acute radiation sickness(ARS).Methods 16 beagles were given 4.5 Gy60 Co γ-rays total body irradiation,and then randomly assigned into irradiation control group,supportive care group and cytokines group.In addition to supportive care,recombinant human granulocyte colony-stimulating factor (rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2(rhIL-2)were administered subcutaneouly to dogs in cytokines group.Peripheral blood hemogram was examined once every two days.Bone marrow and peripheral blood were collected to proceed colony cultivation 4 d pre-irradiation and 1 and 45 d post-irradiation.Conventional histopathological sections of sternum were prepared to observe the histomorphology changes. Results After irradiation,the population of all kinds of cells in peripheral blood declined sharply.WBC nadir Was elevated(1.04×109/L,but 0.28×109/L and 0.68×109/L for the irradiation control group and the supportive care group separately),the duration of thrombocytopenia was shortened (24 days,but 33 days for the supportive care groug) and red blood cell counts were maintained in the range of normal values after cytokincs treatment in combination.The colony forming efficiency of haemopoietic stem cells(HSCs)in bone marrow and peripheral blood decreased obviously 1 d post irradiation,but recovered to the level of that before irradiation 45 d post irradiation after supportive care and cytokines treatment.Hematopoietic cells disappeared in bone marrow of animals in irradiation control group,but hematopoietic functions were recovered after cytokines were administrated.Conclusions RhG-CSF.rhIL-11 and rhIL-2 used in combination could elevate WBC nadir,accelerate the recovery of leukocytes,platelets and red blood cells and promote the proliferation,differentiation and maturity of HSPCs left in the body after 4.5 Gy γ-rays total body irradiation,eventually restore the hematopoietic function.Hence,combination of rhG-CSF,rhIL-11 and rhIL-2 could serve as better therapeutic strategy to treat extremely severe myeloid ARS.     

Therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objectivc To observe the therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy60 Co γ-rays irradiation in beagles,and to provide experimental evidences for the clinical treatment of extremely severe myeloid acute radiation sickness(ARS).Methods 16 beagles were given 4.5 Gy60 Co γ-rays total body irradiation,and then randomly assigned into irradiation control group,supportive care group and cytokines group.In addition to supportive care,recombinant human granulocyte colony-stimulating factor (rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2(rhIL-2)were administered subcutaneouly to dogs in cytokines group.Peripheral blood hemogram was examined once every two days.Bone marrow and peripheral blood were collected to proceed colony cultivation 4 d pre-irradiation and 1 and 45 d post-irradiation.Conventional histopathological sections of sternum were prepared to observe the histomorphology changes. Results After irradiation,the population of all kinds of cells in peripheral blood declined sharply.WBC nadir Was elevated(1.04×109/L,but 0.28×109/L and 0.68×109/L for the irradiation control group and the supportive care group separately),the duration of thrombocytopenia was shortened (24 days,but 33 days for the supportive care groug) and red blood cell counts were maintained in the range of normal values after cytokincs treatment in combination.The colony forming efficiency of haemopoietic stem cells(HSCs)in bone marrow and peripheral blood decreased obviously 1 d post irradiation,but recovered to the level of that before irradiation 45 d post irradiation after supportive care and cytokines treatment.Hematopoietic cells disappeared in bone marrow of animals in irradiation control group,but hematopoietic functions were recovered after cytokines were administrated.Conclusions RhG-CSF.rhIL-11 and rhIL-2 used in combination could elevate WBC nadir,accelerate the recovery of leukocytes,platelets and red blood cells and promote the proliferation,differentiation and maturity of HSPCs left in the body after 4.5 Gy γ-rays total body irradiation,eventually restore the hematopoietic function.Hence,combination of rhG-CSF,rhIL-11 and rhIL-2 could serve as better therapeutic strategy to treat extremely severe myeloid ARS.     

电离辐射致股骨头坏死早期病理特点和机制的研究

Objective To observe the earlier pathological character and mechanism of radiation osteonecrosis in femoral head, in order to provide evidences for the earlier diagnosis and prevention of radiation osteonecrosis of femoral head. Methods Single femoral head of rats were irradiated singly with 30 Gy of 137 Cs γ-ray. The rats were executed after 2, 6 and 12 weeks, then the femurs were stained with HE and histopatholngical changes were observed by light microscope. The bone marrow mesenchymal stem cells (BMSCs) were cultured after 2 weeks and its proliferation and the colony formation were observed. The rats were endo-perfused with microfili contrast medium 12 weeks later, and the 3-dimensional structure of capillaries by Micro-CT was re-estabhshed to detect the pathological changes of capillaries after irradiation. Result The irradiated femur showed deranged cbondrocyte, decreased osteocyte, shrinking nucleus, increased empty bone lacuna and reduced bone trabocnla (P < 0.05). Micro-CT showed the discontinued small vessels and absence(6.65 %) capillaries in irradiated femur were obviously less than those of the unirradiated (12.3 %)(P < 0.001). The proliferation of BMSCs was slowed, the number of colony in irradiated group (10 %) was less than that of control (21 %) (P < 0.001). Conclusions The preliminary histopathological changes of osteoradionecrosis on femoral head could be increased the empty bone lacuna, and the bone lacuna above 30 % was the sign of the earlier period of osteoradionecrosis. The osteonecrosis of femoral head induced by radiation is not only correlated to the damages to the bone, but also to the damages to BMSCs and capillaries.