低能重离子剂量-存活率效应及其拟合模型探讨

低能重离子注入生物体是20世纪80年代中期发展起来的一种诱变手段, 在诱变育种和放射医学等领域有巨大的应用前景。 物理辐照的剂量与存活率之间的关系是辐射生物学效应的最重要的关系之一, 低能离子与生物体相互作用的剂量 存活率曲线表现为特殊的马鞍型。 介绍了低能重离子的马鞍型剂量 存活率效应及其数学模型, 并探讨了模型拟合中存在的一些问题。 Low energy heavy ion implantation is an organism mutation method developed in middle of 1980s, which has a potential application in breeding and radiation therapy. The dose survival curve is a characteristic effect in radiobiology. Implanted with low energy heavy ions, organisms could manifest a Saddle like dose survival curve. In this article, the Saddle like dose survival effect and its modeling has been described, and several topics in model simulation are discussed.     

低能离子诱导拟南芥基因组不稳定性的研究

体细胞同源重组产生的DNA重排、 缺失和复制等是基因组不稳定的重要指标, 以拟南芥菜GUS基因重组报告系R2L100和R3L66为实验材料, 以体细胞同源重组频率（每个植株上的GUS斑点数目）作为评估标准, 研究低能Ar+离子和α粒子辐射对植物基因组稳定性的影响。 结果表明: 30 keV的Ar+离子辐照拟南芥干种子, 在500×1013—3 000×1013 ions/cm2剂量范围内, 同源重组频率与对照相比明显升高, 最大值是对照的2.4倍; 3.3 MeV的α粒子辐照萌发4 d后的幼苗, 同源重组频率随着剂量的增加呈先增后降的变化趋势, 最大值是对照的1.9倍, 对应的辐照剂量是10 Gy。 以上实验结果表明, 低穿透能力的辐射能有效增加植物基因组的不稳定性。 α粒子辐照拟南芥菜幼苗的根, 未受到辐照的地上部分的同源重组频率较对照增加2.5倍, 表明低能离子诱导的基因组不稳定信号在植物个体水平是可以长程输运的。 以上结果从另一个侧面解释了低能离子的诱变机制。 The somatic homologous recombination was frequently used to evaluate genome stability because it can result in DNA changes, such as rearrangement, deletion and duplication. In this paper, we used Arabidopsis thaliana transgenic for GUS recombination substrate (R2L100 and R3L66) to study the genomic instability induced by low energy ion and α particle characteristic of short penetrating properties. The dry seeds of R3L66 line were irradiated by 30 keV Argon ion, the Homologous Recombination Frequency (HRF) had a significant increase at dose range of 500×1013—3 000×1013 ions/cm2. The highest level of HRF was 2.42 fold over the control. The 3.3 MeV α particles were used to radiate 4 day old seedlings of R2L100 line. The HRFs had a dose dependent increase at dose of 1—10 Gy, and a dose dependent decrease at 10—100 Gy. The highest level of HRF induced by α particle was 1.9 fold over control at the dose of 10 Gy. These results indicate that short penetrating irradiation can effectively trigger the plant genomic instability at the level of plant. The local irradiation on the roots of R2L100 by α particle resulted in a 2.5 fold increase of HRF in non irradiated aerial plant,which indicates that a signal of genomic instability generated by α particle radiation can systemically travel in whole plant. It is possible that the genome instability induced by low energy ion is a major part of its mutagenic mechanism.     

碳离子辐射诱导秀丽隐杆线虫生殖细胞凋亡研究

重离子辐射具有独特的深度剂量分布和较高的相对生物学效应,被认为是理想的放疗手段。重离子的生物学效应在径迹形成过程中由多个物理参量共同决定,而这些物理参量和离子入射深度紧密相关,因此明确离子不同入射深度的生物学效应对重离子肿瘤放疗方案的设计和优化有着重要的理论和应用价值。使用兰州重离子研究装置HIRFL-CSRe 终端的碳离子束作为辐射源,以活体模式动物线虫作为实验对象,以线虫生殖细胞的凋亡水平作为生物学检测终点,研究了10 和20 Gy 碳离子辐射在辐射的入口、坪区和峰区的当代生物学效应和对后代个体基因组不稳定性的影响。结果表明:10 和20 Gy 碳离子辐射在三个不同的辐照区域内均显著增加了辐射当代的线虫生殖腺细胞的凋亡水平,并表现出一定的辐射区域和辐射剂量依赖性。同时,辐射诱导的后代个体基因组不稳定性也表现出一定的辐射区域和辐射剂量相关性。Heavy ion irradiation is a perfect means in radio-therapy due to its special depth dose distribution and high relative biological effects. The biological effects of heavy ion irradiation are determined by some major physical parameters, and vary along the tracks of heavy ions. Therefore, it is very significant for the tumor radio-therapy to investigate the biological effects along whole range of heavy ion radiation. In the present study, Caenorhabditis elegans, a model in vivo, was irradiated by carbon ion beams from HCRFL-CSRe, The level of germ cell apoptosis of worms was used as a checking endpoint for DNA damage, the effects of carbon irradiation located in the entrance, plateau and peak regions on the genomic instability of the irradiated worm and their progeny were detected. The results showed that the 10 and 20 Gy of carbon ion radiations led to the increased germ cell apoptosis in irradiated worms and these effects depend on the worm location along the range of carbon ions and the irradiation dosage. The results also suggested that heavy ion irradiation induced the up-regulated genomic instability in their progeny, and might be related to both the irradiation dose and the irradiated location.     

α粒子辐照诱导拟南芥菜早期远程表观遗传改变研究

由于低能离子较低的组织穿透能力, 其诱变机理一直是研究者争论的问题。 近年来, 本研究组的一系列研究工作已经证明在植物中存在辐射远程（诱变）效应, 从一个新的角度解释了低能离子的诱变机理, 然而依然无法解释低能离子辐照中的许多独特生物现象, 而这些现象均具有明显的表观遗传学特性。 以表观遗传学最具标志性特征的胞嘧啶甲基化为研究对象, 以α粒子 拟南芥菜根辐照实验体系作为研究平台, 检测了远程组织(器官)甲基化相关基因 AtDML3 的表达及特定基因片段的甲基化水平。 研究证实, 在植物个体水平辐射可以诱导远程表观遗传的变化, 为进一步探索低能离子的诱变机理提供了新的思路。 Along the way, the mutagenic mechanism of low energy ions irradiation is a debatable issue. Recently, the existence of radiation induced long range (mutagenic) effects in vivo in plants has been performed in a series of studies of our group, which account for the mutagenesis of low energy ions irradiation in a new perspective. However, numerous distinct biology phenomena remain to be addressed, which bear obvious characteristics to epigenetic. In the present study, using the expression of methylation related AtDML3 gene and methylation level of specific gene segments as end points, the methylation of cytosine, the most important feature of epigenetic, was investigated. It was shown that, in A. thaliana, root loca lized α irradiation could induce epigenetic changes in aerial parts which avoided the direct irradiation. The radiation induced long range epigenetic changes were confirmed in this study, which supplied innovative ideas for the further investigation of the mutagenetic mechanism of low energy ions irradiation.     

离子束注入胸腺嘧啶的紫外、红外效应研究

本文中我们利用紫外、红外光谱等分析手段获取低能离子束生物诱变的信息。发现用30 ke V低能氮离子和氩离子在 0 - 2× 1 0 16 ions/cm2注入剂量范围内注入胸腺嘧啶 ,其残存率随剂量的变化趋势均出现了不同于γ射线等辐照时的线性或肩型曲线 ,而呈现一种新的变化趋势——“马鞍型”曲线 ,表明参与结构的质量沉积不足以解释“马鞍型”曲线。红外光谱的分析结果表明在离子束注入过程中胸腺嘧啶分子确实存在结构上的修复。研究结果表明紫外、红外光谱可以给出离子束注入前后生物分子结构突变或重组、分子结构突变与注入剂量的关系以及分子的修复等信息。     

基于大肠杆菌的重离子辐射靶点序列定位和LacI突变分析的实验体系构建及验证

重离子辐射具有独特的物理和生物学特性,在诱变育种等领域有着广泛的应用,但其诱变的机制并不完全清楚。不同于传统的X和γ射线,重离子辐射具有较高的线传能密度(Linear energy transfer,LET),主要诱导团簇状的DNA损伤,其演化为遗传变异的过程更为复杂,突变类型也更难预测。目前的实验技术很难在序列水平对重离子击中DNA的靶点进行定位,这致使重离子辐射诱变机制的研究相对滞后。针对这一问题,根据重离子辐射诱导的团簇损伤核心区域富含DNA双链断裂(Double-strand break,DSB)以及同源重组机制对DSB特异性响应的特性,首先构建了四环素抗性基因(TetA)同源重组元件用于确定DNA团簇损伤的序列定位,并在重组原件侧翼连接反向突变筛选基因LacI用于团簇损伤—突变的检测,最后把该质粒转化到大肠杆菌E.coll。在此基础上,比较分析γ射线与碳重离子(80 MeV/u)辐照后同源重组和报告基因突变的情况,验证了该体系用于重离子辐射靶点序列定位及突变检测的可行性,为进一步研究重离子辐射诱变的相关机制奠定了方法学基础。     

植物个体远程辐射信号早期传递过程的研究体系创建

自2005年以来,活体中的远程辐射旁效应逐渐成为辐射生物学的研究热点,然而其早期信号传递过程的相关研究却鲜见报道,主要原因是由于早期信号传递过程研究所需的旁区与辐射区的自由分离与组合在动物模型上无法实现。本研究是基于植物个体可以切割和嫁接的特性,借鉴离体细胞培养基转移方法研究旁效应早期过程的思想,在植物个体上实现旁区与辐照区的"分离"与"组合",构建了一种研究个体远程辐射信号早期传递过程的植物实验体系。具体是以模式植物拟南芥菜转基因系(AtRAD54promoter∶∶GUS)为材料,同源重组修复相关基因AtRAD54表达水平为生物学检测终点,人为地将辐照区的组织(或器官)与旁区部分"分离"或"嫁接",通过测定旁区组织(或器官)的AtRAD54基因表达水平变化,研究其辐射信号传递的早期过程。该研究体系的创建为活体旁效应早期过程的研究提供了一种可行的研究方法。