微束技术在放射生物学中的应用

微束装置可以为生命科学研究提供微米定位、剂量特定的电离辐射,在生物体内的电离辐射靶物质及其敏感度、靶物质的损伤及修复机制研究中具有独特的作用。概述了生物微束装置和实验技术的发展及其在低剂量辐射效应、旁观者效应、信号传导研究中的主要应用;介绍了中国科学院近代物理研究所（IMP）重离子微束装置,该装置可以提供能量7~ 80 MeV/u、传能线密度为30~ 3000 keV/μm的重离子微束,实现了活细胞辐照和在线观察、小鼠定位辐照的实验技术;利用IMP微束装置在重离子诱导旁效应实验、小鼠下丘脑重离子辐照效应和DNA损伤快速修复动态等方面取得了一些实验成果。The microbeam facility can provide micrometer scale localized and predefined ionizing radiation in the life science study, and the microbeam techniques play a unique role in determining the target substances of ionizing radiation, as well as in the study of radiation sensitivity, mechanisms of radiation damage response and repair. This paper summarizes the technical developments of biological microbeam facilities and their applications in the studies of low-dose radiation effect, bystander effect and cellular signaling. This paper also introduces the recent developments at the heavy-ion microbeam facility in the Institute of Modern Physics (IMP), which can provide heavy ion microbeam irradiation with energy of 7~80 MeV/u and LET of 30~3000 keV/μm. The facility can perform radiobiological irradiation and online investigation in living cells and mice, including bystander effect study, sleeping system influence after irradiation to mice hypothalamus and the recruitment dynamics of XRCC1 protein.     

RADIOACTIVITY AND RADIATION PROTECTION

<正>Revised August 2013 by S.Roesler and M.Silari(CERN).35.1.Definitions[1,2]It would be desirable if legal protection limits could be expressed in directly measurable physical quantities.However,this does not allow to quantify biological effects of the exposure of the human body to ionizing radiation.For this reason,protection limits are expressed in terms of so-called protection quantities which,although calculable,are not measurable.Protection quantities quantify the extent of exposure of the human body to ionizing radiation from both whole and partial body external irradiation and from intakes of radionuclides.In order to demonstrate compliance with dose limits,so-called operational quantities are typically used which aim at providing     

重离子诱导的质粒DNA双链断裂分布研究

利用能量为7．2MeV／u氖离子束辐照体外质粒DNA:pUC18，采用恒场凝胶电泳结合多功能荧光成像系统研究了pUC18双链断裂片段的分布。证实了双链断裂片段分布的非随机性，结果还发现DNA断裂后片段的交联现象，而且交联片段的分布也是非随机的。DNA is considered to be the most important and sensitive target in biological systems. In addition to the base damage, DNA strand breaks are the major lesion in the genome due to exposure to ionizing radiation. Mutation can be introduced to DNA as a result of enzymatic processing of DNA lesions or post irradiation replication. However, the mechanisms of radiation induced mutations are not well clarified at the molecular level. To study the effect on the simple plasmid DNA of heavy ion is even predominant or more feasible. Plasmid pUC18 DNA was prepared and irradiated by neon beam (7.199 MeV/u). The fragment distributions were determined by quantifying the ethidium bromide fluorescence. It can be seen that the shape of the intensity distributions is vastly different for the used radiation Dose. The distribution produced shows an excess of fragments particularly in 3 000 and 10 000 Gy the size range between 20—40 kbp and 20—50 bp. This clustering of double stranded fragments might be influenced by the higher order chromatin structure of genomic DNA. If so, DNA loop structures could correspond to the size range for which we observed DSB clustering. Further studies aim at elucidating the heterogeneity of DSB induction within the genome and investigate the influence of chromatin structure on the non random fragment distribution.     

电离辐射诱导的旁观者效应研究

列举了一些具有代表性的研究结果， 概括介绍了一些产生旁观者效应的可能机制以及辐照引起的活性氧基团或分子、 细胞通讯和细胞因子在旁观者效应中的重要作用. 此外， 还讨论了旁观者效应的表达程度与辐照剂量和射线品质的关系. 旁观者效应的研究结果表明: 辐射产生的潜在危害可能要比以前估计的大， 这给如何评估辐射对人类造成的危害带来了新的冲击. Recent studies have indicated that biological effects, such as chromosomal aberration, gene mutation and cell death and so on, can be induced in cells that are not traversed by radiation directly. This phenomenon has been termed as bystander effects. In this paper, a few representative studies were reported and the possible mechanisms underlying the bystander effects were summarized. The reactive oxygen species (ROS) induced by ionizing radiation, cellular communication and some factors play important roles. Besides, the expression extent of bystander effects depended on radiation dose and quality were discussed. Bystander effects suggest that potential health risks associated with radiation exposure may be greater than those of original thought and this makes ultimate impact on human radiation risk assessment.     

Study on Plasmid and Damage Induced by Low-energy Neon Ion Irradiation

DNA is considered to be the most important and sensitive target in biological systems. Beside base damage, DNA strand breaks are the major lesion in the genome due to exposure to ionizing radiation. Mutation can be introduced to DNA as a result of enzymatic processing of DNA lesions or post-irradiation replication. However, the mechanisms of radiation-induced mutations are not well clarified at the molecular level. A good way to approach the mechanism is to irradiate the plasmid DNA of heavy ion, then transfect the DNA to host cells to determine the mutation spectra. So to study the effect of heavy ions on the simple plasmid DNA is even predominant or more feasible.     

辐照诱导的人正常肝细胞系HL-7702细胞延迟效应

利用X射线辐照人正常肝细胞系HL-7702细胞, 运用胞质分离阻滞微核法实验检测细胞微核率,AnnexinV FITC细胞凋亡检测试剂盒检测细胞凋亡率, 细胞微核率和凋亡率随着辐照剂量的增加而显著增加。X射线照射后细胞传代培养, 第7代时不同剂量辐照后子代细胞微核率和凋亡率同未辐照细胞相比已无明显区别。 对不同剂量辐照后传代7代的细胞再次照射2.5 Gy的相同剂量,发现它们细胞微核率和凋亡率存在明显差异,即初次受辐照剂量高的细胞, 再次以相同剂量辐照后的微核率和凋亡率也高. 这些结果表明,X射线辐照导致了HL-7702细胞基因组不稳定性这一辐射延迟效应,再次辐照使得辐射的延迟效应得以明显的表现。 Human normal liver cell line HL-7702 cells were irradiated with different doses of X rays. Micronucleus and apoptosis rates in the irradiated cells were measured with cytokinesis block micronucleus method and Annexin V FITC apoptosis detection kit, respectively. Experimental data showed that the micronucleus and apoptosis rates increased obviously with increasing irradiation dose. After seven population doublings, the micronucleus and apoptosis rates of the cells surviving exposure to the X rays reduced to the same levels as non irradiated control cells; the progenies of the cells were secondly exposed to X rays at the same dose of 2.5 Gy. We found that the progenies of the cells surviving the first irradiations of the various doses showed markedly differential micronucleus frequencies and apoptotic rates. Although the same dose of 2.5 Gy was applied in the second irradiations, the micronucleus frequencies and apoptotic rates of the progenies of the cells initially exposed at higher doses were significantly higher than the others. These results indicate that X rays lead to genomic instability in HL 7702 cells, which is an important manifestation of radiation induced delayed effect, and a second radiation stimulus makes the delayed effect in the progeny of the previously irradiated cells be expressed obviously.     

12C6+离子对阿维链霉菌累进辐照诱变效应研究

选用12C6+ 离子束对阿维链霉菌诱变选育高产菌株与原始菌株进行辐照诱变, 研究其累进辐照效应。实验结果表明,在辐照剂量为10 Gy时, 原始菌株比诱变高产菌株存活率高, 抗辐射能力强;辐照剂量高于30 Gy时,诱变高产菌株比原始菌株存活率高, 抗辐射能力强。原始菌株正突变率最高的辐照剂量为50 Gy, 致死率99.43%,正突变率最高, 达34.2%;对诱变高产菌株辐照剂量为30 Gy,致死率94.97%,正突变率最高, 达23.5% 。累进辐照效应降低了最佳辐照剂量。 Mutagenic effect on the mutant high producing strain ZJAV Y1 203 and the original strain ZJAV A1 irradiated by ion beam of 12C6+ have been investigated. The experimental results indicated that the original strain has higher survival rate and stronger resistance to radiation than mutant high producing Strain at dose of 10 Gy. The mutant high producing strain has higher survival rate and stronger resistance to radiation than the original strain at the dose higher than 30 Gy. The lethality was 97% and the highest rate of orthomutation was 34.2%, when ZJAV A1 was irradiated by 50 Gy 12C6+ beam. The lethality was 94.97% and the highest rate of orthomutation was 23.5% when ZJAV Y1 203 was irradiated by 30 Gy 12C6+ beam. The best radiation dose is decreased by progressivity irradiation.     

12C6+束辐照引起的实验猪皮肤急性损伤反应的研究

探讨了正常皮肤对重离子辐照急性损伤反应的耐受性, 为重离子治癌临床应用提供安全性检测的实验依据。实验前10 min, 实验猪肌肉注射复方氯胺酮1.2 mg/kg进行麻醉, 然后在兰州重离子研究装置（HIRFL）辐照终端利用12C6+束照射, 辐照剂量分别为0, 12, 21和27 Gy, 辐照分3次完成, 剂量率约为1.2 Gy/min, Bragg峰区照射, 辐照后每隔7 d对照射野拍照并活检取样, 做HE组织病理学观察。不同剂量12C6+离子束辐照实验猪皮肤后, 皮肤外观反应随辐照剂量增大而加快, 表现为肿胀和色素沉积等; 皮肤组织结构的变化明显, 上皮细胞排列紊乱、 萎缩、 空泡变性; 基本恢复正常所需时间也越长, 且都存在明显的剂量效应关系。结果表明, 辐照剂量范围为0—27 Gy时, 重离子对正常皮肤的辐照是安全的。The tolerance of the normal skin to the acute radiation injury reaction induced by heavy ion beams has been studied experimentally. The experimental pigs were injected with 1.2 mg/kg ketamine in 10 min before irradiation and were irradiated with 0, 12 , 21 and 27 Gy 12C6+ ion at a dose rate of 1.2 Gy/min at the Heavy Ion Research Facility in Lanzhou（HIRFL）. The total radiation dose was finished by 3 times at Bragg Peak Region of Heavy Ion Beams.The radiation fields of skin were taken photo and performed biopsy. The contaneous tissues of radiation fields were stained by HE and examined histopatholo gical changes every seven days after irradiation. The results indicated that the cutaneous appearance reaction became more faster with radiation dosage rising and presented with swollen, melanin forming and so on after irradiated by the carbon ions at different dosage. The Pathological examination showed noticeable changes in histological and structural of experimental pigs skin, such as atrophy, vacuolation, denaturation and arranged irregularly in epithelial cells. Furthermore, the time for return to normality became longer with the increasing of radiation dosage. All indexes demonstrated correlation between the does and effects. It is concluded that the irradiation of heavy ion beams to normal skin is security when the radiation dose range is about 0—27 Gy.     

内质网应激对碳离子辐照宫颈癌HeLa 细胞的影响

利用不同剂量的碳离子辐照二硫苏糖醇(2.5 mmol/L) 预处理的HeLa 细胞,探讨了内质网应激反应对碳离子辐照宫颈癌HeLa 细胞的影响。实验发现:与单独辐照组相比,二硫苏糖醇联合碳离子辐照后细胞的存活率下降,而凋亡率增加;二硫苏糖醇联合碳离子辐照加重了碳离子辐照引起的细胞周期阻滞;且联合辐照组的自噬被明显激活。结果表明,持续的内质网应激可改变宫颈癌HeLa 细胞对碳离子辐照反应,且二硫苏糖醇可能通过影响HeLa 细胞的自噬性细胞死亡通路发挥作用。To investigate the effect of endoplasmic reticulum stress on HeLa cells to 12C6+ ion irradiation,HeLa cells were pretreated with 2.5 mmol/L dithiothreitol and irradiated by 12C6+ ions with different doses.The results showed that, compared with IR alone, dithiothreitol combined with carbon ion irradiation caused HeLa cell survival decreased, and the apoptosis increased. Moreover, dithiothreitol and carbon ion radiation combination treatment led to a significant increase of G2/M phase, and autophagy was activated obviously in combination treatment group. The results imply that continuous endoplasmic reticulum stress can change the response of HeLa cells to 12C6+ irradiation, and dithiothreitol may affect HeLa cells through the autophagy cell death pathway.     

长链非编码RNA XR_923426参与调控电离辐射诱导的微丝骨架变化

细胞微丝骨架是多功能亚细胞结构,是电离辐射的感受器也是效应器。为了明确长链非编码RNA(lncRNA)是否参与调控电离辐射引起的微丝骨架动力学变化,采用Swinholide A处理诱导细胞微丝骨架解聚之后,进行lncRNA组学分析并验证差异表达的lncRNA;采用鬼笔环肽染色和微丝网络结构分析等方法评价电离辐射引起微丝骨架动力学变化的程度。结果发现,微丝骨架解聚后,lncRNA XR_923426的表达下调;人为上调lncRNA XR_923426的表达水平能够显著缓解辐射导致的微丝骨架结构异常和微丝网络连接减少。这些发现为微丝通过lncRNA调控辐射导致细胞死亡或肿瘤转移的机制提供了新的研究思路,有望成为肿瘤治疗和正常组织辐射防护的新靶点。     

MOS结构电离辐射效应模型研究

基于氧化层空穴俘获和质子诱导界面陷阱电荷形成物理机制的分析,分别建立了MOS结构电离辐射诱导氧化层陷阱电荷密度、界面陷阱电荷密度与辐射剂量相关性的物理模型.由模型可以得到,在低剂量辐照条件下辐射诱导产生的两种陷阱电荷密度与辐射剂量成线性关系,在中到高辐射剂量下诱导陷阱电荷密度趋于饱和,模型可以很好地描述这两种陷阱电荷与辐射剂量之间的关系.最后讨论了低剂量辐照下,两种辐射诱导陷阱电荷密度之间的关系,认为低辐射剂量下两者存在线性关系,并用实验验证了理论模型的正确性.该模型为辐射环境下MOS器件辐射损伤提供了更 关键词： MOS结构 辐射 界面陷阱 氧化层陷阱     

FT-IR spectroscopy assessment of aesthetic dental materials irradiated with low-dose therapeutic ionizing radiation

The aim of the present study was to evaluate the effects of low-dose therapeutic ionizing radiation on different aesthetic dental materials. Forty five specimens (n = 45) of three different aesthetic restorative materials were prepared and randomly divided into five groups: G1 (control group); G2, G3, G4, G5 experimental groups irradiated respectively with 0.25, 0.50, 0.75, and 1.00 Gy of gamma radiation by the ⁶⁰Co teletherapy machine. Chemical analyses were performed using a FT-IR Nicolet 520 spectrophotometer with reflectance diffuse technique. Even a minimal exposition at ionizing radiation in therapeutic doses can provide chemical changes on light-cured composite resins. The three studied restorative materials showed changes after exposure at gamma radiation, however the increase of the radiation dose did not contribute to an increase in this effect.     

ncRNA-mediated bistability in the synthesis of hundreds of distinct mRNAs and proteins

The kinetics of gene expression can be bistable due to the feedback between the mRNA and protein formation. In eukaryotic cells, the interplay between mRNAs and proteins can be influenced by non-coding RNAs. Some of these RNAs, e.g., microRNAs, may target hundreds of distinct mRNAs. The model presented here shows how a non-coding RNA can be used as a mediator in order to involve numerous mRNAs and proteins into a bistable network.     

Q(γ-H2AX), an analysis method for partial-body radiation exposure using γ-H2AX in nonhuman primate lymphocytes

We previously used the γ-H2AX assay as a biodosimeter for total-body-irradiation (TBI) exposure (γ-rays) in a rhesus macaque (Macaca mulatta) model. Utilizing peripheral blood lymphocytes and plucked hairs, we obtained statistically significant γ-H2AX responses days after total-body exposure to 1-8.5 Gy ((60)Co γ-rays at 55 cGy min(-1)). Here, we introduce a partial-body exposure analysis method, Q(γ-H2AX), which is based on the number of γ-H2AX foci per damaged cells as evident by having one or more γ-H2AX foci per cell. Results from the rhesus monkey - TBI study were used to establish Q(γ-H2AX) dose-response calibration curves to assess acute partial-body exposures. γ-H2AX foci were detected in plucked hairs for several days after in vivo irradiation demonstrating this assay's utility for dose assessment in various body regions. The quantitation of γ-H2AX may provide a robust biodosimeter for analyzing partial body exposures to ionizing radiation in humans.     

Photoluminescence and gamma-ray irradiation of SrAl2O4:Euand Y2O3:Eu phosphors

Y₂O₃:Eu³⁺ phosphor is a very attractive material for use as a red phosphor in many fields. SrAl₂O₄:Eu²⁺ belongs to long lasting phosphor (LLP) and it is a useful bluish-green luminescence material, which can also be a promising candidate as a simple and easy-to-use radiation detection element for visual display of two dimensional radiation distributions. In the present study, both these two kinds of phosphors were synthesized using high temperature solid state reactions. In our work, the influence of gamma-ray irradiation on the properties of these two kinds of phosphors was studied by comparing photoluminescence, brightness and the decay curve of unirradiated and gamma-ray-irradiated samples. Conclusions from the present work can be briefly summarized as follows. In irradiated samples, the brightness is decreased without sensible change in the wavelength distribution of the luminescence spectrum and in the decay kinetic upon gamma exposure. Moreover, the emission due to Eu³⁺→Eu²⁺ conversion in Y₂O₃:Eu³⁺ phosphors was not observed in our sample after irradiation to high exposure. Also the brightness of SrAl₂O₄:Eu²⁺ phosphor turned out to decrease after the exposition to ionizing radiation while the luminescence wavelength distribution remained unchanged. The reason for the effect of gamma-ray irradiation on the properties of phosphors is also discussed in the paper.     

Die Kinetik der Verteilung von 131I nach einer akuten pansomatischen Exposition von 390 mC. kg–1

Es wurde die Verteilung von ¹³¹I 0,5 bis 24 Stunden nach einer einmaligen pansomatischen Exposition mit ionisierender Strahlung von 390 mC. kg^?1 untersucht, wobei das Radioiod 2 und 18 Stunden nach der Ganzkörperbestrahlung verabreicht wurde. Nach der Exposition mit ionisierenden Strahlen sind zeitliche und quantitative Veränderungen der intrathyroidalen und extrathyroidalen Verteilung von Radioiod zu beobachten, was auf eine morphologische und funktionelle Depression der Organe zurückzuführen ist, womit auch eine beschleunigte Eliminierung des verabreichten Radionuklids aus dem Organismus zusammenhängt.

The study involves the tracing of iodine-131 distribution from 0.5 to 24 hours after a pansomatic exposure with an ionizing radiation of 390 mC ? kg^-1. The iodine-131 was administrated at 2 and at 18 hours after the irradiation exposure and was followed in different organs of mice as test animals. The ionizing radiation leads to a temporal and quantitative change of iodine-131 in the intra- and extrathyroidal distribution which is caused by morphological and functional depression of the different organs. This is in turn connected with an accelerated elimination of the radioiodine from the organism.     

利用拉曼光谱技术研究低剂量X射线辐射对人神经母细胞瘤细胞的影响

低剂量电离辐射引发的生物效应复杂而多样,其研究往往又受到辐射标志物和检测技术手段的限制。将拉曼光谱技术应用于低剂量辐射生物效应研究,利用10 mW,532 nm共聚焦拉曼光谱对经过100,200和500 mGy三种辐射剂量的X射线辐照之后的人神经母细胞瘤细胞进行检测,发现细胞嘌呤核苷酸（722～728和1 572～1 581 cm-1等等）、嘧啶核苷酸（770～785 cm-1等等）等DNA相关的拉曼特征峰受到电离辐射影响而发生变化,说明低剂量X射线辐照造成细胞DNA水平改变。采用流式细胞术对同样条件辐照后培养6 h的人神经母细胞瘤细胞进行细胞周期分析发现,三种剂量的X射线电离辐射均造成细胞在G2期阻滞,同样提示电离辐射引起DNA水平升高。通过划痕实验分析辐照后20 h的细胞迁移能力,结果显示,相较于未接受X射线照射的对照细胞,受到三种剂量电离辐射的人神经母细胞瘤细胞均出现迁移水平下降。研究结果表明,通过拉曼光谱分析发现低剂量X射线电离辐射引起人神经母细胞瘤细胞DNA水平变化,其结果与细胞周期分析和迁移分析的结果相一致,但检测时间大大提前,利用拉曼光谱技术可以实现低剂量辐射损伤等细胞生物学效应的早期发现与监测。