乏氧条件下X射线诱导神经胶质母细胞瘤T98G细胞的旁效应

采用条件培养基和共培养处理方式,应用微核形成试验研究人神经胶质母细胞瘤 T98G 细胞,在乏氧条件下经 X 射线诱导的旁效应及其发生机制.研究发现,乏氧条件下,X 射线诱导未照射组细胞的微核率与相应的照射剂量之间存在显著的正相关关系;条件培养基方式下自由基抑制剂二甲基亚砜相似文献   

受辐射肝癌细胞所诱导旁效应与p53的关系

通过检测受照射细胞及与其共培养旁细胞的损伤情况及p53抑制剂对其的影响,研究了肝癌细胞辐射敏感性及辐射诱导的旁效应与p53的关系.发现肝癌细胞的辐射敏感性与p53密切相关:野生型p53辐射敏感性最高,突变型的次之,缺失型的敏感性最低.同时,辐射诱导的旁效应与p53状态亦密切相关,仅野生型p53肝癌细胞(HepG2)对旁细胞(chang氏肝细胞)具有旁效应,且未受照射旁细胞中产生的微核具有明显的剂量效应和时间效应;而突变型(PLC)和缺失型(Hep3B)的肝癌细胞几乎不能诱导辐射旁效应的产生.另外,p53抑制剂可显著抑制辐射旁效应的产生.     

细胞质受照射的遗传损伤效应

通过使用精确单粒子微束装置照射人与中国仓鼠杂交AL细胞细胞质,显示α粒子照射细胞质只有轻微的致死率,照射16个α粒子后仍有76％的细胞可以存活。诱变试验结果发现,尽管单个α粒子照射细胞质在CD59基因位点诱变频率较低,但8个或多于8个α粒子可诱导出高于未受照射对照3倍的诱变频率聚合酶链式反应（PCR）分析发现,照射细胞质诱导的CD59^-基因突变子其11号染色体上Wilms肿瘤（WT）、过氧化氢酶（CAT）、（H－rasRAS)、甲状旁腺激素（PTH）、（脱脂蛋白－A1 APO－A1）等基因缺失率很小,其突变谱类似于自发突变子的突变谱。DMSO或BSO处理细胞的研究显示,自由基清除剂DMSO可降低照射细胞质所诱发的CD59基因突变频率;与此相反,能与细胞内的谷胱甘肽结合的BSO可增加突变频率。结果表明,自由基很可能是照射细胞质诱发突变的主要原因之一。此外,用IF7单克隆抗体进行的8－羟基脱氧鸟苷免疫过氧化物酶染色反应发现照射细胞质可在核内引起DNA的氧化损伤。     

125I籽源持续低剂量率照射诱导人肺癌细胞的旁效应

观察125I籽源持续低剂量率照射诱导人肺癌细胞损伤的旁效应.选择对高剂量率外照射敏感性不同的A549人肺腺癌细胞和NCI-H446人小细胞肺癌细胞,采用125I籽源离体照射细胞模式,将直接受照细胞与未受照细胞共培养24h,应用CB微核法和γH2AX荧光免疫分析法,检测2Gy和4Gy125I籽源持续低剂量率照射诱导人肺癌细胞的微核形成和DNA双链断裂水平.结果表明,125I籽源照射能显著诱导A549和NCI-H446细胞的微核形成率和γH2AX位点形成率增加的旁效应,显示增强对肿瘤细胞的杀伤作用.旁效应强弱与累积照射剂量、肿瘤细胞的辐射敏感性相关:对高剂量率外照射敏感的NCI-H446细胞对低剂量率照射及其旁效应的敏感性高于A549细胞,但与直接辐射效应相比,125I籽源照射诱导A549细胞旁效应高于NCI-H446细胞,这两种细胞的辐射旁效应均随累积照射剂量增加而降低,提示介导旁效应的信号因子水平可能随细胞损伤程度的增加而下降.     

CB法微核检测技术应用于诱导基因组不稳定性的研究

对γ射线诱导中国仓鼠成纤维(CHL)细胞基因组不稳定性进行了研究。利用CB法微核检测技术对一次照射、二次照射的CHL细胞进行微核测定,观察DNA损伤程度的变化情况。结果显示,微核发生率与细胞受照剂量呈相关关系,并随培养时间延长呈下降趋势。细胞再次受照后,其微核发生率与一次受照剂量呈相关关系。表明微核发生率与染色体损伤程度存在剂量相关性,微核检测技术可以用于生物效应表现为染色体损伤的辐射诱导基因组不稳定性的研究。     

α粒子照射诱发细胞基因突变的旁效应及机理研究

对α粒子照射诱发人类11号染色体(Hchr 11)基因突变的旁效应及其可能的机理进行研究。用包含单条Hchr 11的人一中国仓鼠卵巢细胞杂交细胞系(A1)为靶细胞,经CD59表面抗原抗体在补体存在下筛选突变细胞克隆,测定Hchr 11基因突变率;在α粒子照射源与受照射细胞间插入网格,定比例照射细胞,观察α粒子照射的细胞对周围未受照射细胞基因突变的影响,即旁效应;通过观察自由基清除剂二甲基亚砜(DMSO)和细胞间通讯阻断剂高丙体六六六(Lindane)对α粒子照射诱发Hchr 11基因突变旁效应的抑制作用探讨其可能的机理。单纯α子照射细胞诱发的基因突变率与照射剂量存在明确的剂量效应关系;用α粒子加网照射的实验模型,仅15％的细胞受到照射时,群体细胞的基因突变率明显高于受照射细胞的预期基因突变率,表明未受照射的细胞中也发生了基因突变;DMSO能显著减少α粒子照射细胞诱发的基因突变,但对其诱发的基因突变旁效应无明显抑制作用;与此相反,Lindane对单纯α粒子照射细胞诱发的基因突变无明显影响,但能显著降低α粒子照射细胞诱发的基因突变旁效应。α粒子照射细胞诱发的基因突变存在旁效应;细胞间通讯在α粒子照射诱发细胞基因突变旁效应中起重要作用。     

a粒子照射诱发细胞存活的旁效应及机理研究

以人-中国仓鼠卵巢杂交细胞（AL细胞）为靶，通过在照射源与受照射细胞间插入网格对细胞进行定比照射，以及用受照射细胞培养液培养未受照射细胞两种方式，检测未受照射细胞存活率变化，研究a粒子照射细胞时对未受照射细胞存活的影响。通过观察自由基清除剂二甲基亚砚（DMSO）和细胞间通讯阻断剂Lindane对a粒子照射诱发的细胞存活旁效应的抑制作用探讨其可能的机理。研究结果表明，a粒子照射细胞时的未受照射细胞，以及受a粒子照射细胞的培养液培养的未受照射细胞，其存活率均明显低于预期或正常细胞水平；而DMSO和Lindane均能明显提高细胞存活率。这提示a粒子照射细胞存在旁效应，活性氧和细胞间通讯在a粒子诱发细胞存活旁效应中可能起着重要作用。     

高LET的7Li离子致DNA损伤的直接和间接作用研究

在HI-13串列加速器加速的具有高LET值的7Li离子辐照不同浓度的pUC19质粒DNA水溶液、加自由基清除剂(甘露醇)的DNA水溶液以及干状DNA样品,利用高分辨的原子力显微镜技术,研究7Li致DNA损伤的直接作用和间接作用.结果显示,在相同剂量下,7Li离子比低LET辐射能诱发更多的双链断裂,形成更多的集团损伤,使DSB的分布更局部和更密集.对于水溶液DNA,7Li离子的水辐解产生的自由基的间接作用在DNA分子链断裂的产生方面发挥着重要作用,而且自由基清除剂甘露醇能有效地保护DNA分子.     

培基介导的辐射旁效应及机理初探

采用ICR小鼠,转移受照射细胞的条件培养液来培养未受照射细胞,检测YAC—I（小鼠T淋巴瘤）细胞对小鼠自然杀伤（NK）细胞的敏感性变化,测定小鼠脾细胞增殖能力,观察自由基清除剂二甲基亚砜（Dimethyl sulfoxide,DMSO）照射前处理细胞的影响。初步研究了电离辐射旁效应（Bystander effect,BE）及其可能的机理。结果显示,条件培养液培养的Yac—I细胞对NK细胞的敏感性明显升高。用条件培养液培养的小鼠脾细胞的增殖能力也明显受到抑制,提示辐射旁效应的存在,观察到DMSO对旁效应有一定的抑制作用,说明活性氧（Reactive oxygen species,ROS）等自由基对旁效应有一定贡献。然而在淋巴细胞增殖实验ICM＋1％DMSO组的数据显示DMSO对旁效应的抑制作用非常有限,表明除自由基外必定存在其他因素介导旁效应的发生。     

碳离子辐照哺乳动物细胞引起的旁观者效应

用经过碳离子辐照V79细胞的条件培养基(Irradiated conditionedmedium,简称ICM)和与受照射细胞共同培养两种方法,研究了未受照射细胞的旁观者效应。结果表明,ICM法可以明显降低未受照射细胞的克隆形成率;受照射细胞与未受照射细胞共同培养一段时间后,细胞克隆形成率比假定没有旁观者效应时的预期值低,细胞微核率和hprt(次黄嘌呤磷酸核糖基转移酶)基因位点突变率与预期值基本相同。推测受照射细胞可能释放出了能对未受照射细胞生长产生毒性的因子,这种因子对未受照射细胞没有明显的致微核和致突变作用。     

Differential modulation of a radiation-induced bystander effect in glioblastoma cells by pifithrin-α and wortmannin

The implication of radiation-induced bystander effect (RIBE) for both radiation protection and radiotherapy has attracted significant attention, but a key question is how to modulate the RIBE. The present study found that, when a fraction of glioblastoma cells in T98G population were individually targeted with precise helium particles through their nucleus, micronucleus (MN) were induced and its yield increased non-linearly with radiation dose. After co-culturing with irradiated cells, additional MN could be induced in the non-irradiated bystander cells and its yield was independent of irradiation dose, giving direct evidence of a RIBE. Further results showed that the RIBE could be eliminated by pifithrin-α (p53 inhibitor) but enhanced by wortmannin (PI3K inhibitor). Moreover, it was found that nitric oxide (NO) contributed to this RIBE, and the levels of NO of both irradiated cells and bystander cells could be extensively diminished by pifithrin-α but insignificantly reduced by wortmannin. Our results indicate that RIBE can be modulated by p53 and PI3K through a NO-dependent and NO-independent pathway, respectively.     

α粒子照射诱发细胞存活的旁效应及机理研究

以人－中国仓鼠卵巢杂交细胞（AL细胞）为靶，通过在照射源与受照射细胞间插入网络对细胞进行定比照射，以及用受照射细胞培养液培养未受照射细胞两种方式，检测未受照射细胞存活率变化，研究α粒子照射细胞时对未受照射细胞存活的影响。通过观察自由基清除剂二甲基亚砜（DMSO）和细胞间通讯阻断剂Lindane对α粒子照射诱发的细胞存活旁效应的抑制作用探讨其可能的机理。研究结果表明，α粒子照射细胞时的未受照射细胞，以及受α粒子照射细胞的培养液培养的未受照射细胞，其存活率均明显低于预期值或正常细胞水平；而DMSO和Lindane均能明显提高细胞存活率。这提示α粒子照射细胞存在旁效应，活性氧和细胞间通讯在α粒子诱发细胞存活旁效应中可能起着重要作用。     

Bystander effect in human hepatoma HepG2 cells caused by medium transfers at different times after high-LET carbon ion irradiation

Although radiation-induced bystander effects have been well documented in a variety of biological systems, whether irradiated cells have the ability to generate bystander signaling persistently is still unclear and the clinical relevance of bystander effects in radiotherapy remains to be elucidated. This study examines tumor cellular bystander response to autologous medium from cell culture irradiated with high-linear energy transfer (LET) heavy ions at a therapeutically relevant dose in terms of clonogenic cell survival. In vitro experiments were performed using human hepatoma HepG2 cell line exposed to 100 keV/μm carbon ions at a dose of 2 Gy. Two different periods (2 and 12 h) after irradiation, irradiated cell conditioned medium (ICCM) and replenished fresh medium were harvested and then transferred to unirradiated bystander cells. Cellular bystander responses were measured with the different medium transfer protocols. Significant higher survival fractions of unirradiated cells receiving the media from the irradiated cultures at the different times post-irradiation than those of the control were observed. Even replenishing fresh medium for unirradiated cells which had been exposed to the ICCM for 12 h could not prevent the bystander cells from the increased survival fraction. These results suggest that the irradiated cells could release unidentified signal factor(s), which induced the increase in survival fraction for the unirradiated bystander cells, into the media sustainedly and the carbon ions triggered a cascade of signaling events in the irradiated cells rather than secreting the soluble signal factor(s) just at a short period after irradiation. Based on the observations in this study, the importance of bystander effect in clinical radiotherapy was discussed and incorporating the bystander effect into the current radiobiological models, which are applicable to heavy ion radiotherapy, is needed urgently.     

H1299旁效应细胞对X-射线辐射的适应性与TGF-β1通路相关

本研究探索辐射诱导人非小细胞肺癌H1299旁效应细胞的适应性反应及TGF-β1通路在其中的作用。采用培养液转移法得到辐射诱导的旁效应细胞,用克隆形成实验检测旁效应细胞受照射后的适应性反应,用Western Blotting检测旁效应细胞中SOD2的表达变化。结果发现:用条件培养液培养H1299旁效应细胞,并不影响细胞的克隆存活率;但是细胞经1 h和18 h未照射条件培养液培养后再接受2Gy的X-射线照射,其细胞存活率较培养于新鲜培养液的细胞受照后的存活率分别增加了12%和38%,经1 h和18 h照射条件培养液培养后的细胞再接受2Gy的X-射线照射,其细胞存活力在此基础上又分别增加了10%;当在照前用TGF-βR1抑制剂SB431542处理信号细胞后,旁效应细胞的适应性反应不再发生;而将SB431542直接加入条件培养液中,并未影响1 h条件培养液诱导的适应性,但是用含有SB431542的18 h未照射和照射条件培养液培养过的旁效应细胞经2 Gy照射后,其克隆存活率较未加SB431542组分别降低了28%和18%,18 h未照射和照射条件培养液组间差异却增大至24%;旁效应细胞经照射条件培养液培养3 h或5 h后其SOD2表达下降。以上结果表明经条件培养液培养后旁效应细胞对X-射线照射产生了适应性反应,照射条件培养液与未照射条件培养液相比,进一步增加了这种适应性,并且TGF-β1信号通路对该适应性有重要调控作用,而SOD2可能未参与这个过程。     

Bystander effects induced by medium from carbon-ion irradiated human cancer cells

The bystander effects induced by medium from human hepatoma SMMC-7721 and adenocarcinoma F56 cells irradiated with carbon ions were investigated. It was found that the survival fraction （SF） of the irradiated cells decreased exponentially along with the increased dose. SMMC-7721 cells were more radiosensitive than F56 cells. The plating efficiency （PE） of the non-irradiated cells treated with irradiated conditioned medium （ICM） was obviously lower than the PE of control cells for SMMC-7721 cells but not for F56 cells. Moreover, the reduced PE and SF by ICM treatment were more significant for IGy irradiation than for 6Gy irradiation on SMMC-7721 cells, These results suggest that the irradiated cells can secrete factor（s） into medium that is cytotoxic to bystander non-irradiated cells. The bystander effects are dependent on cell genotype presented at the time of irradiation and radiation dose, This makes impact on the precise estimation of the effects of radiation and tumor radiotherapy,     

Cell Locating with the Image Analysis System of the CAS-LIBB Single-Particle Microbeam Facility

A single-particle microbeam facility has been constructed at the Key Laboratory ofIon Beam Bioengineering (LIBB), Chinese Academy of Sciences (CAS). At the CAS-LIBB microbeam facility, we have developed protocols to place exact numbers of charged particles through nuclear centroids of cells, at defined positions in the cytoplasm relative to the nucleus, and through defined fractions of cells in a population. In this paper, we address the methods for nucleus, cytoplasm and bystander (either a single or an exact number of ions is delivered to a certain percentage of cells in a population to study the bystander effects of radiation) irradiation in detail from theprecision of target finding and cell locating in the image analysis system. Moreover, for cells touching slightly in an image, a watershed method is used to separate these touching objects;after that, the number of objects in an image is counted accurately and the irradiation points are located precisely.     

极低频磁场联合或不联合X射线对CHO细胞动粒阳性和阴性微核生成的影响

Extremely low frequency magnetic field (ELFMF) produced by power lines and household electric appliances has been associated with increased incidence of cancers, as was suggested by several epidemiological studies. To test the genotoxic effects of ELFMF, the induction of micronuclei by exposure to ELFMF and/or X-rays was investigated by cytokinesis-block method in cultured Chinese Hamster Ovary (CHO) cells.     

Helium-implanted CLAM steel and evolutionary behavior of defects investigated by positron-annihilation spectroscopy

China Low Activation Martensitic (CLAM) steel has been chosen as the primary candidate structural material for the first wall/blanket for fusion reactor. The excessive helium irradiation induced damage of CLAM steel at high temperatures and the evolution of defects were investigated in this paper. The samples were homogeneously implanted with 1e + 17 ions/cm² and 100 keV of helium at room temperature, 473, 673, and 873 K. Irradiation induced damage of CLAM steel and the annealing behavior of defects were probed by slow positron beam Doppler broadening technique. Helium implantation produced a large number of vacancy-type defects which bound with helium and formed helium–vacancy complexes. Target atoms’ displacement capacity was strengthened with rising irradiation temperatures, so the S parameter increased with increasing irradiation temperatures, and helium–vacancy complexes were main defects after helium implantation at damage layers. Helium bubbles would be unstable and the desorption of helium bubbles would promote the density of defects above 673 K. By analyzing the curves of S–W and annealing tests of irradiated specimen, it suggested that there werenot only one type of defect in damage layers. Though helium–vacancy complexes were primary defects after helium implanted, introducing excessive helium might also generated other point defects or dislocation loops in the material.