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.     

X射线致HeLa细胞损伤过程中NADPH氧化酶的作用

以HeLa细胞为实验材料, 探讨了NADPH氧化酶在X射线诱导细胞损伤过程中的作用。 结果显示, 12 Gy X射线辐照后细胞内活性氧(ROS)明显增加, 在用NADPH氧化酶抑制剂处理后再辐照, 则细胞内ROS降低到未辐照水平; 同时辐照后NADPH 氧化酶细胞质亚基p47phox 在细胞质积聚并和细胞膜亚基 gp91phox 结合; Western blotting检测结果显示, NADPH 氧化酶的关键亚基 gp91phox 的表达量明显增加。 以上结果说明, NADPH氧化酶可以被X射线激活, 由其介导产生的ROS在X射线诱导HeLa细胞损伤过程中扮演重要角色。 To investigate the role of NADPH oxidase in HeLa cell lesion induced by X ray irradiation, the change of cell survival was detected with MTT assay, reactive oxygen species (ROS) was measured by fluorospectrophotometer. Immunostaining and confocal laser scanning microscopy was employed to detect the co localization of two subunit of NADPH oxidase, p47phox and gp91phox in the cell. Western blotting was used to detect the expression of gp91phox before and after X ray irradiation. After X ray irradiation, intra cellular level of ROS increased obviously. But the increase could be blocked by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase. Meanwhile, cytosolic subunit p47phox moved to membrane and co localizated with gp91phox after irradiation. Moreover, the results also show that gp91phox increased sharply after 12 Gy X ray irradiation. Therefore, NADPH oxidase mediated production of ROS plays an important role in HeLa cell lesion induced by X ray.     

真空、低能离子注入对人宫颈癌（HeLa）细胞中 p53及c-fos mRNA的表达影响

在具有高效护水功能石蜡油的保护下进行了人宫颈癌细胞(HeLa细胞)的低能离子注入及真空处理, 用荧光定量PCR的方法分别研究了真空和30 keV N+的不同注入剂量下, 细胞中p53基因和c-fos基因的mRNA表达变化。 结果表明, 上述两个基因的表达与真空及低能离子注入之间存在着剂量关系。     

NO分子双光子诱导荧光的自吸收现象

以皮秒Nd:YAG激光器抽运的光学参量发生/放大器为激发光源,获得了在452.4 nm波长激光激发下,NO分子的双光子激光诱导色散荧光光谱(LIDFS)。并由此得到了NO分子基电子态的振动基频和振动非谐性系数分别为ω″_e=(1 904.7±7.3) cm-1, ω″_eχ″_e=(14.2±1.2) cm-1, ω″ey″_e=-(0.021 8±0.009 1) cm-1。在NO分子双光子LIDFS实验研究中,首次观测到了NO分子强烈的自吸收现象,在较高样品气压条件下,色散荧光光谱图中对应A 2Σ(v′＝0)→X 2Π(v″＝0)跃迁的谱线消失。实验结果表明,自吸收现象导致的该跃迁谱线强度的变化,随样品气压、激光场和样品作用区到荧光接收窗口距离的减小而减小。     

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.     

ROS enhancement by silicon nanoparticles in X-ray irradiated aqueous suspensions and in glioma C6 cells

The capability of silicon nanoparticles to increase the yield of reactive species upon 4 MeV X-ray irradiation of aqueous suspensions and C6 glioma cell cultures was investigated. ROS generation was detected and quantified using several specific probes. The particles were characterized by FTIR, XPS, TEM, DLS, luminescence, and adsorption spectroscopy before and after irradiation to evaluate the effect of high energy radiation on their structure. The total concentration of O₂ ^•−/HO₂ ^•, HO^•, and H₂O₂ generated upon 4-MeV X-ray irradiation of 6.4 μM silicon nanoparticle aqueous suspensions were on the order of 10 μM per Gy, ten times higher than that obtained in similar experiments but in the absence of particles. Cytotoxic ¹O₂ was generated only in irradiation experiments containing the particles. The particle surface became oxidized to SiO₂ and the luminescence yield reduced with the irradiation dose. Changes in the surface morphology did not affect, within the experimental error, the yields of ROS generated per Gy. X-ray irradiation of glioma C6 cell cultures with incorporated silicon nanoparticles showed a marked production of ROS proportional to the radiation dose received. In the absence of nanoparticles, the cells showed no irradiation-enhanced ROS generation. The obtained results indicate that silicon nanoparticles of <5 nm size have the potential to be used as radiosensitizers for improving the outcomes of cancer radiotherapy. Their capability of producing ¹O₂ upon X-ray irradiation opens novel approaches in the design of therapy strategies.     

曲古菌素A作用下HeLa细胞的吸收光谱分析

用紫外分光光度计采集了人宫颈癌细胞经不同剂量的曲古菌素A（TSA）作用24h和48h后的吸收光谱。结果表明：细胞在260nm附近存在特征性的吸收峰,且200nmol/L的药物剂量会使HeLa细胞在260nm处的吸收值最大。该项工作找到了细胞经药物处理后吸收光谱与用药剂量之间的关系,为药物的筛选提供了新的思路。