辐射诱发细胞凋亡中DNA大片段的生成

目的与方法：ＤＮＡ在核小体间断裂,琼脂糖凝胶电泳时出现梯形谱（ＤＮＡｌａｄｄｅｒ）通常被认为是细胞凋亡的特征性生化标志,利用脉冲场凝胶电泳,对人外周血淋巴细胞受照后ＤＮＡ梯形谱形成前,ＤＮＡ断裂情况进行了观察。结果人外周血淋巴细胞受照者凋亡过程中,ＤＮＡ的断裂分成两步,首先是产生３００ｋｂ到５０ｋｂ的ＤＮＡ大片段,随后才是通常所描述的ＤＮＡ梯形谱。结论;细胞凋亡中核酸内切酶对ＤＮＡ的降解不是一步完     

X射线对小鼠胸腺细胞凋亡的影响

采用荧光分光光度法对裂解ＤＮＡ进行定量，并用琼脂糖凝胶电泳法定性研究Ｘ射线全身照射后小鼠胸腺细胞凋亡。结果表明，４ＧｙＸ射线照后２小时胸腺细胞凋亡开始增加，于照后１４小时达峰值，尔后呈下降趋势。ＤＮＡ裂解率在照后２４小时降至１４小时的５０％，这可能是内环境中巨噬细胞吞噬凋亡小体所致。在照射后１４小时研究其剂量－效应关系发现，高剂量照射使ＤＮＡ裂解明显增多，形成１８０ｂｐ（ｂａｓｅｐａｉｒｓ）左右或其整倍数的ＤＮＡ断片，电泳呈现＂梯形图谱＂；而低剂量辐射可使ＤＮＡ裂解率降低。剂量－效应曲线呈Ｊ型。     

^60Co辐射事故5名受照者外周血T细胞抗原受体基因的…

在低熔点琼脂糖胶内制备人淋巴细胞基因组ＤＮＡ，两种限制性核苷酸内切酶对低熔点胶块内的样本ＤＮＡ进行完全消化，以人Ｔ淋巴细胞抗原受体（ＴＣＲ）的α和β链ｃＤＮＡ为探针，对５名４．５年前全身受＾６０Ｃｏ大剂量受照者外周血淋巴细胞基因组ＤＮＡ进行印迹杂交分析。结果发现经ＨｉｎｄⅢ内切酶消化的样本ＤＮＡ，正常对照与受照者比较，两种ｃＤＮＡ探针的杂交结果无明显区别，经ＥｃｏＲＩ内切酶消化的ＤＮＡ样本，４名受     

60Co辐射事故5名受照者外周血T细胞抗原受体基因的印迹杂交分析

在低熔点琼脂糖胶内制备人淋巴细胞基因组ＤＮＡ，两种限制性核苷酸内切酶对低熔点胶块内的样本ＤＮＡ进行完全消化，以人Ｔ淋巴细胞抗原受体（ＴＣＲ）的α和β链ｃＤＮＡ为探针，对５名４．５年前全身受６０Ｃｏ大剂量受照者外周血淋巴细胞基因组ＤＮＡ进行印迹杂交分析。结果发现经ＨｉｎｄⅢ内切酶消化的样本ＤＮＡ，正常对照与受照者比较，两种ｃＤＮＡ探针的杂交结果无明显区别，经ＥｃｏＲⅠ内切酶消化的ＤＮＡ样本，４名受者的αｃＤＮＡ探针杂交带型不同于正常对照。作者对这一结果进行了初步分析。     

Okadaic acid诱导早熟凝集染色体测定辐射生物剂量

目的建立人外周血的早熟凝集染色体（prematurely condensed chromosomes，PCC）环与辐射剂量之间的剂量．效应关系曲线。方法取健康成年人外周血，^60Coγ射线分别以0、1、2、5、10、15、20和25Gy照射，吸收剂量率为1Gy／min，培养48h，终止培养前1h加入Okadaic acid诱导早熟凝集染色体，观察人外周血淋巴细胞PCC环与剂量效应的关系。结果Okadaic acid诱导的人外周血淋巴细胞早熟凝集染色体环随着辐射剂量的增加而增加，但在20Gy以后达到饱和。结论在20Gy剂量范围内，PCC环与辐射剂量具有良好的剂量．效应关系，与双着丝粒相比，它可作为大剂量受照情况下的生物剂量指示剂。     

357例放射工作人员外周血淋巴细胞微核分析

对淋巴细胞微核率的观察，不仅能早期发现辐射效应，而且还能对受照个体进行生物剂量估算。随着电离辐射装置的改进及辐射防护措施的加强，放射工作人员受照剂量逐年降低。为探讨低剂量电离辐射对放射工作人员细胞遗传学的影响，笔者于2003年11—12月对濮阳市放射工作人员进行了外周血淋巴细胞微核分析，结果报道如下。     

小鼠受亚致死剂量60Co γ 射线照射后胸腺细胞凋亡的研究

目的探讨小鼠受亚致死剂量６０Ｃｏγ射线照射后胸腺细胞凋亡的变化规律，为研究辐射后免疫功能的重建打下基础。方法采用６０Ｃｏγ射线，２．０，４．０，６．０Ｇｙ单次全身照射，用ＰＩ染色流式细胞仪分析，ＤＮＡ琼脂糖凝胶电泳及细胞激活增殖能力的测定等方法观察胸腺细胞凋亡的规律。结果胸腺细胞照后２小时即出现明显的细胞凋亡，４～８小时达高峰，后渐减少，４．０Ｇｙ，６．０Ｇｙ在３６小时内的凋亡率均比２Ｇｙ高，照后１０天三个剂量组的凋亡率基本接近正常；ＤＮＡ琼脂糖凝胶电泳有大量的小分子片断，呈典型的梯状图谱；４Ｇｙ组用ＰＨＡ、ｒＩＬ－２不同组合刺激受照组细胞，发现胸腺细胞凋亡率有不同程度的下降。结论亚致死剂量６０Ｃｏγ射线照射后小鼠胸腺细胞出现明显的凋亡，４Ｇｙ照射剂量可能是胸腺细胞达凋亡高峰的最适剂量，ＰＨＡ、ｒＩＬ－２联合对辐射诱导的胸腺细胞的凋亡有明显的保护作用。     

应用玫瑰花结形成试验测定人淋巴细胞的辐射敏感性

作者应用玫瑰花结形成试验的方法观察了人淋巴细胞各亚群的辐射敏感性,试图寻找更敏感的生物剂量测定指标。方法:血液样品采自11名21～25岁的男性供血者。应用葡聚糖-泛影葡胺梯度密度离心的方法从外周血中分离淋巴细胞。细胞悬液分为两部分,一部分照射X线,另一部分作生物对照。照射剂量为100～2500伦。照后2、4、24、48、72小时进行检查。予定照后     

5例放射事故病人照后5.5年外周血淋巴细胞 HPRT 基因突变频率和突变谱的测定

通过克隆外周血淋巴细胞并用６巯基鸟嘌呤筛选次黄嘌呤磷酸核糖转移酶基因（ｈｙｐｏｘａｎｔｈｉｎｅｐｈｏｓｐｈｏｒｉｂｏｓｙｌｔｒａｎｓｆｅｒａｓｅ，ＨＰＲＴ）缺陷的突变细胞，测定了５例急性放射病人在受２．０～５．２Ｇｙ６０Ｃｏγ射线意外照射后５．５年外周血淋巴细胞ＨＰＲＴ基因突变频率。观察到细胞克隆效率、突变细胞克隆数、ＨＰＲＴ突变频率与受照剂量有一定的依赖性。用多引物聚合酶链反应（ＰＣＲ）扩增突变细胞ＨＰＲＴ基因全部９个外显子伴以琼脂糖凝胶电泳以分析基因突变谱。结果表明，高剂量受照病人的外显子缺失总数较低剂量者高。与照后３．５年和４．５年的突变谱比较，外显子缺失突变比例逐年下降，而点突变比例逐年升高。可能反映病人机体的恢复和受损ＤＮＡ的修复。     

重离子辐射对人外周血T淋巴细胞生物学性能的影响

目的研究重离子对人外周血T淋巴细胞增殖、凋亡等生物学性能的影响并探讨其机制,为肿瘤放射治疗的辐射防护提供实验依据和基础。方法 Ficoll分离法分离人外周血T淋巴细胞,采用12C重离子束坪区照射,照射样品能量为70 MeV、LET=29 keV/μm,照射剂量为1.0和2.0 Gy,剂量率为0.5 Gy/min。分别于照射后12、24h,RT-PCR检测凋亡相关基因Bcl-2,Bax,Caspase3,Caspase8和Caspase9的表达;于照射后24、48 h,CCK8法检测细胞增殖能力;于照射后24、48 h,采用AnnexinV-PE/7-AAD、AnnexinV-FITC/PI法检测凋亡发生,并采用RT-PCR检测凋亡相关蛋白Bcl-2、Bax和Caspase3的表达。结果重离子照射可明显抑制人外周血T淋巴细胞的增殖,随着剂量增大,抑制作用更加明显。同时,重离子照射可促进T淋巴细胞的凋亡,特别是对于晚期凋亡的诱导作用（P〈0.01）。RT-PCR检测结果显示,重离子辐射可抑制抗凋亡蛋白Bcl-2的表达,促进促凋亡蛋白Bax和Caspase3的表达（P〈0.01）。结论重离子辐射可显著影响抑制T淋巴细胞的增殖并促进其凋亡。     

Post-irradiation Defects in Newly Synthesized DNA: Neutrons and Electrons Compared and the Effect of Misonidazole

The role of cellular membranes in thymocyte apoptosis has been examined. Trolox, a water soluble analogue of vitamin E and inhibitor of membrane damage, inhibits DNA fragmentative in thymocytes exposed to γ-radiation. Trolox is most effective in inhibiting DNA fragmentation when added to cells within 30 min post-irradiation. Exposure to trolox only during irradiation did not prevent DNA fragmentation, suggesting that it does not work by scavenging free radicals generated during radiation exposure. Incubation of the irradiated cell suspension with trolox for 2 h post-irradiation was sufficient to prevent DNA fragmentation measured at 24 h in irradiated cells. This suggests that trolox irreversibly inhibits a cellular lesion required for apoptosis. The induction of DNA fragmentation appears to be related to a concurrent, pronounced flow of Ca²⁺ into the cell. At 3 h post-irradiation the amount of Ca²⁺ in irradiated thymocytes was more than twice that of unirradiated thymocytes. Membrane damage has been shown to affect the transport of Ca²⁺. Trolox treatment completely blocked the radiation-induced influx of Ca²⁺ into the thymocytes. These results suggest that membrane damage is a critical lesion that is involved in DNA fragmentation in thymocyte apoptosis.     

Accumulation of cAMP in gamma-irradiated thymocytes and internucleosomal DNA fragmentation.

Ionizing radiation, glucocorticosteroids and chemical inducers of differentiation (CID) are cytotoxic to thymocytes, and induce internucleosomal DNA fragmentation. Tissue cAMP levels in thymi of irradiated mice were significantly elevated as early as 30 min post-irradiation. In contrast, cAMP content in the liver was not changed significantly up to 1 h post-irradiation, and then some decrease occurred. Irradiation of isolated thymocytes gave essentially the same results as after irradiation of animals, and the elevation in cAMP 30 min after the irradiation, DNA fragmentation and cell death were linearly related to the dose up to 2.5 Gy. The maximal induction of cAMP level occurs in the fractions of radiosensitive cortical thymocytes. In thymocytes all CID tested also induced the increase in cAMP level with concomitant DNA fragmentation. Unlike ionizing radiation, UVC light did not induce cAMP accumulation and DNA fragmentation in thymocytes. Treatment of UV-irradiated cells with But2 cAMP did not result in an increase in DNA fragmentation. Ionizing radiation induced DNA fragmentation and cell death can be prevented by adding the protein kinases inhibitor H-7. Theophylline was shown to reduce the cAMP response, DNA fragmentation and cell death in gamma-irradiated thymocytes, suggesting that the accumulation of cAMP may be partly related to adenosine receptor sites.     

DNA damage in cultured skin microvascular endothelial cells exposed to gamma rays and treated by the combination pentoxifylline and alpha-tocopherol

PURPOSE: This in vitro study aims at evaluating the effect of the combination of pentoxifylline (PTX) and trolox (Tx), the water-soluble analogue of alpha-tocopherol, on the oxidative state and DNA damage in dermal microvascular endothelial cells exposed to doses up to 10 Gy of ionizing radiation. MATERIALS AND METHODS: Confluent primary cultures of dermal endothelial cells were gamma irradiated at 3 and 10 Gy, and 0.5 mM of both drugs, PTX and Tx, was added either before (15 min) or after (30 min or 24 h) irradiation. Reactive oxygen species (ROS), measured by the dichlorodihydrofluorescein diacetate assay, and DNA damage, assessed by the comet and micronucleus assays, were measured at different times after exposure (0 - 21 days). RESULTS: The PTX/Tx treatment decreased the early and delayed peak of ROS production by a factor of 2.8 in 10 Gy-irradiated cells immediately after irradiation and the basal level by a factor of 2 in non-irradiated control cells. Moreover, the level of DNA strand breaks, as measured by the comet assay, was shown to be reduced by half immediately after irradiation when the PTX/Tx treatment was added 15 min before irradiation. However, unexpectedly, it was decreased to a similar extent when the drugs were added 30 min after radiation exposure. This reduction was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei (MN) frequency observed on days 10 and 14 post-irradiation, respectively. CONCLUSION: These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased MN frequency in 10 Gy-irradiated cells. Thus, these drugs may cause a possible interference with DNA repair processes.     

DNA damage in cultured skin microvascular endothelial cells exposed to gamma rays and treated by the combination pentoxifylline and α-tocopherol

Purpose: This in vitro study aims at evaluating the effect of the combination of pentoxifylline (PTX) and trolox (Tx), the water-soluble analogue of α-tocopherol, on the oxidative state and DNA damage in dermal microvascular endothelial cells exposed to doses up to 10 Gy of ionizing radiation.

Materials and methods: Confluent primary cultures of dermal endothelial cells were gamma irradiated at 3 and 10 Gy, and 0.5 mM of both drugs, PTX and Tx, was added either before (15 min) or after (30 min or 24 h) irradiation. Reactive oxygen species (ROS), measured by the dichlorodihydrofluorescein diacetate assay, and DNA damage, assessed by the comet and micronucleus assays, were measured at different times after exposure (0 – 21 days).

Results: The PTX/Tx treatment decreased the early and delayed peak of ROS production by a factor of 2.8 in 10 Gy-irradiated cells immediately after irradiation and the basal level by a factor of 2 in non-irradiated control cells. Moreover, the level of DNA strand breaks, as measured by the comet assay, was shown to be reduced by half immediately after irradiation when the PTX/Tx treatment was added 15 min before irradiation. However, unexpectedly, it was decreased to a similar extent when the drugs were added 30 min after radiation exposure. This reduction was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei (MN) frequency observed on days 10 and 14 post-irradiation, respectively.

Conclusion: These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased MN frequency in 10 Gy-irradiated cells. Thus, these drugs may cause a possible interference with DNA repair processes.     

Ascorbic acid reduced mutagenicity at the HPRT locus in CHO cells against thermal neutron radiation.

We investigated the biological effects of the long-lived radicals induced following neutron irradiation. It has been reported that radiation-induced long-lived radicals were scavenged by post-irradiation treatment of ascorbic acid (Koyama, 1998). We studied the effects of ascorbic acid acting as a long-lived radical scavenger on cell killing and mutagenicity in Chinese hamster ovary cells against thermal neutrons produced at the Kyoto University Research reactor. Ascorbic acid was added to cells 30 min after neutron irradiation and removed 150 min after irradiation. The biological end point of cell survival was measured by colony formation assay. The mutagenicity was measured by the mutant frequency in the HPRT locus. The post-irradiation treatment of ascorbic acid did not alter the cell killing effect of neutron radiation. However, the mutagenicity was decreased, especially when the cells were irradiated with boron. Our results suggested that ascorbic acid scavenged long-lived radicals effectively and caused apparent protective effects against mutagenicity of boron neutron capture therapy.     

Epstein-Barr virus-transformed lymphoblastoid cell lines of ataxia telangiectasia patients are defective in X-ray-induced apoptosis.

PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) obtained from unaffected healthy individuals and ataxia telangectasia (A-T) patients to undergo apoptosis after X-ray exposure. MATERIAL AND METHODS: The LCL were exposed to 1-4 Gy X-rays at a dose-rate of 1.36 Gy/min. At various post-irradiation times (0, 24, 48 and 72 h) the induction of apoptosis was analysed by: (1) monitoring the formation of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization of apoptotic cells after fluorescence staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, was investigated in these cells. RESULTS: The LCL obtained from the A-T homozygotes were resistant to undergoing radiation-induced apoptosis during the observation time used. On the contrary, LCL from unaffected healthy controls displayed significant radiation-induced chromatin fragmentation seen at 48 h and 72 h after irradiation. In these cells, radiation-induced G -arrest (24h post-irradiation) preceded chromatin cleavage. In A-T LCL, the defective G1-arrest was not followed by apoptosis. CONCLUSIONS: In spite of a defective cell-cycle control, EBV-transformed LCL of A-T patients compared with unaffected healthy controls do not undergo X-ray-induced apoptosis, at least during their first post-irradiation cell cycle.     

The effect of post-irradiation anisotonic treatment on cell survival and repair of DNA damage

V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.     

Reply to Letter by C. B. Seymour and C. Mothersill

Summary

V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.     

60Co γ射线诱导正常人淋巴母细胞PIG3 基因mRNA表达的剂量相关性研究

目的 探讨正常人淋巴母细胞AHH1电离辐射作用后PIG3基因mRNA表达变化的剂量-效应规律,建立一种基于基因表达变化的新型辐射生物剂量计的可能性。方法 激光共聚焦检测DNA双链断裂分子标记γ-H2AX集簇点,1、2、4、6、8和10 Gy ⁶⁰Co γ射线照射AHH1细胞后4、10和24 h收集细胞,应用实时荧光定量PCR技术对PIG3基因mRNA表达水平进行相对定量检测。结果 γ射线照射后30 min检测发现PIG3蛋白与γ-H2AX在细胞核内有部分共定位,形成集簇点(foci),表明其参与电离辐射致DNA双链断裂损伤信号识别反应。γ射线诱导PIG3基因 mRNA表达水平随时间推移不断提高,持续时间可达24 h。PIG3基因mRNA表达水平具有显著的剂量依赖性,其表达丰度变化的剂量依赖范围在照射后4 h为0~ 6 Gy、照射后10和24 h均为0~10 Gy。结论 PIG3基因参与DNA双链断裂损伤信号识别反应,其mRNA的辐射诱导表达具有良好的剂量-效应关系,具有发展成为新的放射生物剂量计的价值。     

Attenuation of caspase-3-dependent apoptosis by Trolox post-treatment of X-irradiated MOLT-4 cells

PURPOSE: The relationship between post-irradiation treatment with Trolox, an antioxidant that inhibits lipid peroxidation, and X-ray-induced apoptosis, with regard to signal transduction pathways, was examined in MOLT-4, a human leukaemia cell line. MATERIALS AND METHODS: In MOLT-4 cells treated with Trolox after X-irradiation, viability, DNA fragmentation, expression of p53, BCL-2, BAX, active SAPK/JNK, active caspase-3 and the cleavage of PARP were measured by the trypan blue exclusion test, agarose gel electrophoresis and Western blotting. RESULTS: Stained cells and ladder-like DNA cleavage were observed after X-irradiation. Cell death and DNA fragmentation were significantly inhibited by the post-irradiation treatment with Trolox. The expression of p53 and active SAPK/JNK was increased after X-irradiation, and fragments of PARP and the activated fragment of caspase-3 were produced. Post-irradiation treatment with Trolox attenuated the X-irradiation-induced expression, fragmentation or activation of these apoptosis-related biomolecules. The expression of BCL-2 and BAX, which would occur downstream from p53, was not changed by irradiation and Trolox treatment. Furthermore, cell death was associated with caspase-3 because the ladder-like DNA cleavage was completely inhibited by Ac-DEVD-CHO but not Ac-YVAD-CHO, TLCK and PMSF. CONCLUSION: Post-irradiation events such as membrane damage induce caspase-3-dependent apoptosis, which might be mediated by the activation of SAPK/JNK and be independent of p53.