C离子辐照引起细胞分泌旁效应信号因子浓度随时间的变化

信号因子通常能介导旁效应的发生。 利用高LET C离子辐照体外培养的人肝癌QGY-7703细胞, 检测辐照后不同时刻培养基中信号因子TGF-β1和NO的浓度, 并通过转移培养基法检测照射后不同时刻转移培养基对人肝L02旁细胞存活率和代谢活力的影响, 发现受照射细胞在时间与空间上调控着周围信号因子的浓度, 并且通过信号因子浓度的变化影响旁细胞的各种效应发生。 实验为旁效应的解释提供了新的实验数据。 Signaling factors usually play an important role in bystander effect. In this work, human hepatoma QGY 7703 cells in vitro were irradiated with high LET carbon ions. Concentrations of signaling factors such as TGF-β1 and NO were measured in the media of the irradiated QGY-7703 cells at different time points after irradiation. The conditioned media harvested at various times post irradiation were transferred to human hepatocyte L02 cells as bystander cells and then the influence of the conditioned media on survival fraction and cell viability of the bystander cells were determined. The results show that the irradiated cells regulate the concentration of the signaling factors released nearby themselves temporally and spatially, and the bystander cells response to the signaling factors differentially according to the concentration change. This work provides new basic data for exploring the bystander effect, especially caused by high LET radiation.     

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.     

羧甲基-β-1,3-葡聚糖对人肝癌HepG2细胞的放射增敏作用

本研究旨在探讨羧甲基-β-1,3葡聚糖（CMG）对人肝癌HepG2细胞X射线或12C6+离子束辐射敏感性的影响。首先用CCK-8法检测CMG对HepG2细胞的生长抑制情况,得到半数抑制浓度（IC50）为120.6μg/mL。用浓度为0.1×IC50的CMG预处理HepG2细胞24 h,再给予2 Gy X射线或12C6+离子束辐照（CMG+辐照组）;CMG未处理组直接接受2 Gy X射线或12C6+离子束辐照（辐照组）。对比分析辐照组和CMG+辐照组细胞的克隆存活、DNA损伤、凋亡与周期分布、细胞内活性氧（ROS）水平。发现:与X射线辐照组相比,相同剂量的12C6+离子辐照组克隆存活率更小,DNA损伤和周期阻滞更加严重,细胞凋亡率和细胞内ROS水平也更高。与单独X射线或12C6+离子束辐照组相比,CMG+辐照组克隆存活率明显降低,细胞凋亡率随辐照后CMG作用时间的延长而明显增加,CMG使辐照后细胞内ROS维持在一个较高的水平,同时CMG明显加重了单独辐照诱导的DNA损伤和周期阻滞。结果表明,与X射线相比,HepG2细胞对相同剂量的12C6+离子辐射更敏感;CMG可增加HepG2细胞对X射线或12C6+离子辐射的敏感性;CMG可能通过增加受照HepG2细胞内的ROS水平,加剧辐照诱导的DNA损伤,促进辐射诱导细胞凋亡而起到辐射增敏作用。This study aims to investigate the effect of carboxymethy-β-1, 3-glucan (CMG) on the sensitivity of human hepatoma HepG2 cells to X-rays or 12C6+ ions irradiation. First, the inhibitory effect of CMG on the growth of HepG2 cells was detected by CCK-8 assay, and the half maximal inhibitory concentration (IC50) was 120.6 μg/mL. HepG2 cells were pretreated with CMG at a concentration of 0.1×IC50 for 24 h and then irradiated with 2 Gy X-ray or 12C6+ ion beams (CMG + irradiation group). CMG untreated group was directly irradiated by 2 Gy X-rays or 12C6+ ions beam (irradiation group). The clone survival, DNA damage, cell apoptosis, cell cycle distribution, and intracellular reactive oxygen species (ROS) levels in irradiation group and CMG + irradiation group were comparatively analyzed. The results showed that the clone survival rate was lower, DNA damage and cycle arrest were more serious, and the rate of apoptosis and intracellular ROS levels were higher in 12C6+ ions irradiation group than those in the same dose of X-rays irradiation group. Compared with X-rays or 12C6+ ions irradiation group, the clone survival rate of CMG + irradiation group was significantly decreased, and the apoptosis rate significantly increased with the prolongation of CMG treatment post-irradiation; CMG maintained intracellular ROS at a higher level after irradiation, CMG also significantly aggravated radiation-induced DNA damage and cycle arrest. These results indicated that HepG2 cells were more sensitive to 12C6+ ions radiation than those at the same dose of X-rays. CMG increased the sensitivity of HepG2 cells to X-rays or 12C6+ ions irradiation by increasing intracellular ROS level, exacerbating radiation-induced DNA damage and promoting radiation-induced apoptosis in irradiated HepG2 cells.     

X射线诱导的HeLa细胞旁效应中ROS和NO关系研究

活性氧(ROS)和一氧化氮（NO)是辐射诱导的旁效应信号通路中的两个重要信号分子。 实验研究了这两种信号分子在HeLa细胞旁效应信号通路中的关系。 通过微核实验, 发现X射线辐照过的HeLa细胞及其旁观者细胞微核形成明显增加, 而二甲亚砜（DMSO）预处理显著抑制了微核形成。 另外还发现, 接受条件培养基的旁观者细胞的增殖速率增加, 而DMSO预处理产生条件培养基的受辐照细胞则使旁观者细胞的增殖速率降低。 以上的结果从不同角度证实了HeLa细胞存在X射线诱导的旁效应, 且其可以被DMSO预处理所抑制。Western blotting和DAF FM DA荧光探针检测分别显示出辐照后细胞的诱导型一氧化氮合酶（iNOS）和NO水平均升高, 而DMSO预处理则降低其水平。 因此, 可以推测X射线诱导的HeLa旁效应当中ROS是NO的上游信号。 Accumulating evidence indicates that irradiated cells can release signals which induce a series of biological responses in non exposed cells. This is known as irradiation induced bystander effects. Both reactive oxygen species(ROS) and nitric oxide(NO) play important roles in bystander effects. In this study, we determined the relationship of ROS and NO in the signaling pathway of bystander effects. HeLa cells were treated with or without dimethye sulfoxide(DMSO) before X ray irradiation, and micronuclei formation as well as cell proliferation rate was detected in both irradiated and bystander cells. In addition, we also detected inducible nitric oxide synthase(iNOS) expression and NO level in irradiated cells using Western blotting and DAF FM DA fluorescent probe, respectively. Our results showed that micronuclei were induced in irradiated and bystander cells while DMSO treatment significantly suppressed the formation of micronuclei in both of them. We also found that when cells were irradiated their proliferation rate was suppressed while DMSO treatment eliminated this inhibition effect. In contrast, the cells received conditioned medium from irradiated cells proliferated more quickly than the cells received medium from non irradiated cells while DMSO treatment reduced the difference. Finally, we found that irradiated cells had higher level of iNOS and NO compared to non irradiated controls, whereas DMSO treatment decreased their levels. These results suggest that ROS is the upstream signal of NO in X ray induced bystander effects in HeLa cells.     

和厚朴酚对非小细胞肺癌细胞的辐射增敏效应

研究了和厚朴酚（HNK）对非小细胞肺癌（NSCLC）细胞系A549和H1299对低线性能量转移（LET） X射线和高LET碳离子的辐射增敏效应。首先用CCK-8检测了HNK对A549和H1299细胞的生长抑制情况,发现20 μmol/L的HNK处理对细胞的生长抑制作用较弱。用该浓度HNK预处理细胞2 h后给予不同剂量X射线或碳离子的照射,克隆存活法检测细胞的辐射敏感性,Annexin-PI双染法检测细胞凋亡,γH2AX焦点法检测DNA的双链断裂（DSB）损伤。实验结果显示:与X射线相比,NSCLC细胞对碳离子更敏感,HNK预处理仅对碳离子照射有辐射增敏作用;与碳离子单独照射相比,HNK预处理联合碳离子照射诱导了更明显的细胞凋亡;在照射后24 h,HNK预处理联合碳离子照射引起的细胞γH2AX焦点阳性率维持在较高水平,而X射线照射没有这些效应。实验结果表明,HNK预处理抑制了NSCLC细胞DNA的DSB修复,诱导了细胞凋亡的发生,从而提高了细胞对碳离子的辐射敏感性。The radiosensitizing effect of Honokiol (HNK) on non-small cell lung carcinoma (NSCLC) cell lines A549 and H1299 to low-linear energy transfer (LET) X-rays and high-LET carbon ions was investigated in this study. First, the inhibitory effects of HNK on the growth of A549 and H1299 cells were detected by CCK-8 assay, and 20 μmol/L HNK treatment was found to induce a growth inhibitory effect slightly in these two cell lines. Cells were pre-treated with HNK and then irradiated with X-rays and carbon ions of different doses. Cellular radiosensitivity, apoptosis and DNA damage were analyzed by clonogenic survival, Annexin-PI staining and γH2AX foci, respectively. The results showed the cells were more sensitive to carbon ion irradiation compared to X-rays and the radiosensitization of HNK was only observed after carbon ion irradiation. Furthermore, the co-treatment led to higher apoptosis rate 48 h after irradiation and increased the positive rate of γH2AX foci 24 h after irradiation in A549 and H1299 cells compared with those in the groups treated with carbon ion irradiation alone. These phenomena were not observed after X-ray irradiation. Our data suggest that the pre-treatment with HNK inhibited DNA DSB repair, induced apoptosis and then enhanced the cellular radiosensitivity to carbon ions in NSCLC cells.     

不同LET射线对细胞的生物学效应

以人肝癌细胞系和正常肝细胞系为材料,报道了不同传能线密度射线辐射引发细胞染色体原初断裂及24 h内的修复情况。 计算了相对生物学效应的值。 以L02染色体总断裂数量得出的RBE值96.05 keV/μm的12C6+ 为3.6, 512 keV/μm 36Ar18+ 为2.9。 而以7721染色体总断裂数量得出的RBE值: 96.05 keV/μm的12C6+ 为3.5,512keV/μm 36Ar18+也为2.9。用产生等点染色单体断裂计算,则RBE更高。对比得出,高LET对增加等点染色单体断裂量的作用要远远大于对增加染色单体断裂量的作用。等点染色单体的断裂修复难度要远远大于染色单体断裂的修复难度, 这也是高LET高致死率的一个重要原因。 Human hepatoma SMMC 7721 and normal liver L02 cells were irradiated with γ rays,12C6+ and 36Ar18+ ion beams at the Heavy Ion Research Facility in Lanzhou(HIRFL). We reported the kinetic repair of chromosome breaks of L02 and SMMC 7721 cells in 24 h of post irradiation time. The relative biological effectiveness(RBE) for inducing chromatid breaks were 3.6 for L02 and 3.5 for SMMC 7721 cell lines at the linear energy transfer(LET) peak of 96.55 keV/μm 12C6+ ions, and 2.9 (both of the two cell lines) at 512 keV/μm 36Ar18+ ions.It suggested that the RBE of isochromatid type breaks induced by 36Ar18+ was higher than those by 12C6+. We concluded that the high production of isochromatid type breaks, induced by the densely ionizing track structure, could be regarded as a signature of high LET radiation exposure.     

辐射诱发体内旁效应特征的研究进展

大量的细胞生物学研究显示, 辐射诱发旁效应的传输信号包括活性氧自由基（ROS）、 一氧化氮自由基（NO）以及一些细胞因子。 近年来,越来越多的文献报道关于体内旁效应特征的研究, 并已经证实在生物体内辐射诱导的旁效应不仅能发生在相邻组织, 还可发生在远源器官。这些体内旁效应包括DNA损伤、表观遗传学改变、miRNA及基因表达改变、 细胞增殖和凋亡等。 为了总结和分析辐射诱发体内旁效应的特征, 本文综述了辐射诱发体内旁效应的特点, 包括其与性别、 传播途径、辐射品质的关系以及相关机制的研究。 Radiation induced bystander effect is defined as the induction of damage in neighboring non hit cells by signals released from directly irradiated cells. ROS, NO and cytokines are involved in signaling pathways of bystander effects. Recently, the bystander effects in vivo have been reported more and more. It has been indicated that radiation induced bystander effect was localized not only in bystander tissues but also in distant organs. This effect includes many biological endpoints such as DNA damage, epigenetic, miRNA and gene expression changes, cell proliferation and apoptosis. In this review we described different aspects of ionizing radiation induced bystander effects such as its characteristics, sex specific, signaling transfer, dose and LET dependence, and related mechanisms.     

Atomic Force Microscopy Measurement of DNA Fragment Induced by Heavy Ions

Choosing ^7 Li and ^12C heavy ions respectively with different linear energy transfer (LET) values, purified plasmid DNA samples in aqueous solution are irradiated with various doses. The atomic force microscopy (AFM) is used for analysis of DNA fragments induced by both the kinds of heavy ions. There is a change of three forms of DNA, i.e. supercoiled, open circular and linear form, as the dose is observed. The distribution function of DNA fragment length is obtained for the first time and fitted with the Tsallis entropy statistical theory. The result indicates that AFM is a useful tool for analysis of the short fragment of DNA, high-LET heavy ion radiation induces DNA double strand breaks (DSBs) more effectively, and the distributions of the DSBs are more local and dense in comparison with low-LET radiation.     

金纳米粒子的放射增敏效应研究

系统阐述了与金纳米粒子(GNPs) 放射增敏效应相关实验的方法与结果、影响GNPs 放射增敏的因素、GNPs 放射增敏的细胞和动物实验表现及其相关机制。同时结合相关实验,分析和比较了15 nm 柠檬酸钠包被的GNPs 的放射增敏效应,发现GNPs 在高LET 的碳离子束和低LET 的X射线辐照下对Hela细胞的杀伤力随其浓度的增加而增大;在50% 的细胞存活率下,当GNPs 的质量浓度为7.5 g/mL时,其X射线的剂量减少率和碳离子的相对生物学效应值(RBE) 的增加率达到了最大,分别为65.3% 和43.6%,同时GNPs 共培养细胞24 和48 h 后,未出现细胞周期同步化的现象。This paper describes the methods and results of the previous experiments, the experimental phenomena of the cell and animal tests and the relative mechanisms on the radiosensitizing effect of GNPs. Together with our experiments, the radiosensitizing effects of 15 nm citrate-capped GNPs and related mechanisms are analyzed and compared, finding that Hela cell killing of GNPs increase along with their concentration after exposure to high- and low-LET radiation such as carbon ions and X-rays. In addition, the percentages of dose reduction of the X-rays and RBE increment of the carbon ions reached their maximums 65.3% and 43.6%, respectively,at 50% survival level when Hela cells were pre-treated with 7.5 g/mL GNPs. Moreover, Hela cells showed no cell-cycle synchronization after 24 and 48 h exposure to GNPs.     

Survivin表达在高LET射线诱导细胞凋亡中

先前的研究表明, 肿瘤细胞中survivin的高表达与细胞对高传能线密度(LET)射线的辐射抗性相关。 研究了survivin表达在高LET射线诱导的细胞凋亡中的作用, 发现抑制survivin表达对高LET C离子辐射诱导的Bcl-2和Bax表达没有明显的影响。 在高LET射线辐照中, survivin可能通过抑制caspase-3和-9活性的途径, 抑制了细胞凋亡。It has been proven that over expression of survivin in cancerous cell lines is related to the radioresistance of cells to high LET radiation in previous work. In this study, action mechanisms of survivin gene in apoptosis induced by high LET radiation were investigated. We found that inhibiting survivin by siRNA had no notable influence on Bcl-2 and Bax expressions induced by carbon ions. Survivin depressed cell apoptosis through the inhibition of the activities of caspase-3 and -9 possibly in cell apoptosis induced by high LET radiation.     

重离子诱导细胞衰老研究

综述了细胞衰老诱导因素和衰老细胞的特征,提出细胞衰老在癌症发生发展过程中具有双重作用效应。同时,以X射线和12C6+离子束对黑色素瘤细胞系92-1或人正常成纤维细胞系MRC5进行辐照,观察分析DNA损伤应答效应和细胞衰老诱导。实验结果表明,相对于X射线,12C6+离子束能导致DNA团簇损伤,持续激活DNA损伤应答,更容易诱导细胞衰老;12C6+离子束能同时诱导癌细胞和正常细胞衰老。这些实验结果暗示开展12C6+离子束诱导细胞衰老研究具有紧迫性。     

12C6+离子预辐射对小鼠胸腺脾脏细胞周期进程的影响

以0, 0.05, 0.1, 0.25, 0.5 Gy 12C6+ 离子全身预辐照昆明小鼠, 间隔4 h后再对小鼠进行4 Gy全身辐射。 辐照后12 h用流式细胞仪检测小鼠胸腺脾脏细胞在各细胞周期时相的百分率, 同时用单细胞电泳检测受辐射小鼠胸腺脾脏细胞DNA损伤程度。 结果显示, 相对于大剂量预照射组, 各低剂量预照射组胸腺细胞S期细胞百分率显著减少; 脾脏细胞G0/G1期细胞百分率明显减少; 同时胸腺脾脏细胞的拖尾率及拖尾长度明显减少, 以0.1 Gy预辐照效果最为明显。 这些结果表明, 低剂量预辐射处理可以减弱胸腺细胞的S期阻滞及脾脏细胞的G1期阻滞, 并明显减轻胸腺脾脏细胞的DNA损伤程度。     

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.     

碳离子辐照对神经胶质瘤干细胞杀伤的研究

虽然重离子束治癌已经被证明有着射程精确、入口坪区剂量小、相对生物学效应高等显著优点,但重离子辐照对肿瘤干细胞所产生的辐射生物学效应特性依旧不明确。本研究使用人源神经胶质瘤干细胞来研究在面对肿瘤干细胞时,重离子相对于传统X射线是否有明显的生物学优势。实验结果证明,在神经胶质瘤干细胞中,2 Gy碳离子造成的DNA损伤的修复率比X射线造成的损伤修复率要低;MTT实验则证明经过碳离子辐照的肿瘤干细胞活力要比X射线辐照的肿瘤干细胞低得多。综上所述,面对神经胶质瘤干细胞,碳离子能更有效地靶向肿瘤干细胞从而相对于传统X射线有明显的生物学优势。这些发现对于更好地理解重离子束治癌相关的生物学效应有重要的作用。Though heavy-ion therapy has demonstrated significant benefits such as well-defined range, small entrance dose and high relative biological effectiveness, the characteristics of radio-biological effects on cancer stem cells induced by heavy-ion treatment is not completely clear. In this paper, we used human glioma cancer stem cells to investigate whether heavy ions offered a biological advantage, by effectively targeting cancer stem cells, in comparison to conventional X-rays. Our results showed that the repair rate of DNA damage generated by 2 Gy of carbon ions was lower than that generated by X-rays in glioma stem cells. MTT assay showed that the viability of cancer stem cells irradiated by carbon ions was significant lower than that irradiated by X-rays. Taken together, carbon ions showed a biological advantage over X-rays by effectively targeting glioma cancer stem cells. These findings have significant importance in understanding the biological effects related to heavy-ion therapy.