线虫辐射损伤信号空间转导与机制研究

发布时间：2018-08-12 19:44

【摘要】：长期以来,人们一直认为辐射的损伤效应仅仅存在于被辐照的细胞,而随着辐射旁效应的发现,在非辐照细胞出现与照射细胞类似的生物学效应,对传统的辐射致癌危险性的线性模型以及辐射防护措施提出新的挑战。辐射旁效应成为目前国际上辐射生物学研究领域的热点之一,涉及到辐射损伤信号的产生、传导和响应,以及信号的级联放大过程。目前,尽管辐射旁效应的现象已经被明确,但是对于辐射损伤信号在不同组织中传递的差异还不清楚。相关的信号转导途径是如何参与到辐射旁效应的过程中的,也还没有被阐明。因此,发展新的辐射技术,将研究体系从离体细胞转移到活体水平,在更为精确的条件下分析和研究低剂量辐射的生物学反应,解析其重要的生物学机制,对于辐射防护和肿瘤治疗具有重要意义。本研究基于中国科学院离子束生物学重点实验室的单粒子微束装置和模式生物秀丽隐杆线虫,定点辐射线虫的消化器官后食道球中心和生殖器官阴门部位,研究了辐射旁效应信号在生殖系统内部传递和从消化系统向生殖系统传递的区别,并探索其内在的分子机制。本论文的主要研究结果如下:1.通过CAS-LIBB单粒子微束装置以3.2 MeV的质子对线虫的咽部后食道球中心和阴门部位进行定点定量辐照,结果表明,系统内和系统间的辐射都可以导致辐射旁效应,与辐射食道球的结果相比,对线虫的阴门部位进行定点辐射时,生殖腺细胞凋亡数目更加显著,说明辐射旁效应信号更容易在生殖系统内部传递。不同的组织对辐射表现出不同的敏感性,因此导致了辐射诱导旁效应的差别。并且辐射损伤信号在生殖系统内部转导会引发更严重的遗传损伤效应,导致线虫生殖腺细胞的基因组不稳定性。2.通过对HUS-1::CFP荧光报告品系的应用,我们观察到定点辐射咽部和阴门部位后,旁区生殖细胞中荧光聚点的产生,直接证明了活体水平辐射旁效应导致的DNA损伤的发生。与此同时,对荧光聚点数量的统计显示了两种类型的辐射旁效应导致的损伤程度的区别,即生殖系统内部的辐射旁效应比从消化系统到生殖系统这种系统间的辐射旁效应导致的损伤要更加严重,这与其他的生物学检测终点所反映的结论也是一致的。线虫单基因突变品系hus-1(op241、cep-1(w40)、egl-1(n487)、ced-4(nU162)、ced-3(n717)的使用和全身性 RNA 干扰mrt-2、hus-1、cep-1、ced-4这四个基因,证明DDR途径对于辐射诱导的旁区生殖腺细胞调亡来说是必不可少的。3.为了进一步确定DDR途径的空间功能,我们利用rrf-1和ppw-1线虫突变品系对DDR途径的关键基因进行组织特异性RNA干扰。结果表明,mrt-2/hus-1/cep-1/ced-4主要在辐射旁效应的响应阶段起作用。与系统内辐射损伤信号转导不需要体细胞内的DDR成分相比,在辐射损伤信号从后食道球向生殖细胞传递时,体细胞组织中的DNA损伤检查点MRT-2和HUS-1对系统间辐射导致的生殖细胞凋亡起到部分作用。但总体来看,DDR途径主要还是在辐射旁效应的响应阶段起作用的。4.为了研究辐射后线虫全身的ROS表达水平,我们使用了 GFP标记的线粒体锰超氧化物歧化酶SOD-3表达和线粒体的解折叠蛋白反应的两个荧光报告品系CF1553和SJ4100,以及分子探针H2DCF-DA。结果显示两个位点的辐射都能导致线虫全身性ROS水平的上升,辐射位点的咽部和阴门部位可以观察到局部ROS增强,而通过自由基猝灭剂DMSO处理,可以恢复旁区生殖细胞的凋亡水平,表明氧化损伤在辐射旁效应的转导中起关键作用。5.初步证明了该过程涉及神经酰胺的参与以及神经酰胺与DDR通路的关联。神经酰胺合酶功能的缺失能有效抑制辐射旁效应诱导的生殖细胞凋亡,因为无论是lagr-1(gk327和hyl-1(ok976)基因单突变,还是lagr-1(gk327),hyl-1(ok976)基因双突变品系,线虫的生殖腺细胞凋亡都被明显抑制,而sphk-1(0k1097)基因突变品系并没有导致生殖腺细胞凋亡发生明显的变化,表明神经酰胺生物合成通路参与了线虫辐射诱导旁效应的诱导。在对神经酰胺代谢合酶基因功能缺失后egl-1/ced-13基因mRNA水平表达量的变化进行探究,结果表明神经酰胺和CEP-1可能平行地对辐射诱导的生殖腺细胞凋亡起协同作用。
[Abstract]:For a long time, it has been thought that radiation damage effects exist only in irradiated cells. With the discovery of radiation side effects, biological effects similar to those of irradiated cells appear in non-irradiated cells, which pose new challenges to traditional linear models of radiation-induced cancer risk and radiation protection measures. One of the hotspots in the field of radiation biology is the generation, transmission and response of radiation damage signals, and the cascade amplification of signals. Therefore, the development of new radiation technologies, the transfer of research systems from cells in vitro to living levels, the analysis and study of biological responses to low-dose radiation under more precise conditions, and the analysis of their important biological mechanisms for radiation protection and cancer treatment, have not been clarified. This study was based on the single-particle microbeam system and the model organism Cryptococcus elegans in the Key Laboratory of Ion Beam Biology, Chinese Academy of Sciences. The signal transduction from the digestive system to the genital system was studied at the posterior esophageal bulb center and the vaginal hilum of the genital organs. The main results of this paper are as follows: 1. Cas-LIBB single-particle microbeam irradiation with 3.2 MeV protons on the posterior esophageal sphere and the vagina of the nematode pharynx was carried out. The results showed that both intra-system and inter-system radiation could cause radiation para-radiation. The number of apoptotic cells in the genital glands of nematodes exposed to fixed-point irradiation was more significant than that of the esophageal bulb irradiation, suggesting that bystander effect signals were more easily transmitted within the reproductive system. Transduction of radiation damage signals in the reproductive system can lead to more serious genetic damage, leading to genomic instability in the gonad cells of nematodes. 2. By applying HUS-1:: CFP fluorescent reporter strain, we observed the production of fluorescent aggregates in the germ cells of the parapharynx and the vagina after fixed-point radiation, which directly proved the existence of fluorescent aggregates in living cells. At the same time, statistics on the number of fluorescent foci show the difference in the degree of damage caused by two types of radiation-induced side effects, i.e. the radiation-induced side effects in the reproductive system are more severe than those caused by the radiation-induced side effects between the digestive and reproductive systems. This is also consistent with other biological endpoints. The use of single-gene mutations hus-1 (op241, CEP-1 (w40), egl-1 (n487), CED-4 (nU162), CED-3 (n717) and systemic RNA interference with mrt-2, hus-1, cep-1, CED-4 in nematode prove that the DDR pathway is involved in radiation-induced paragonadal apoptosis. 3. In order to further determine the spatial function of DDR pathway, we used rrf-1 and ppw-1 nematode mutant strains to interfere with the key genes of DDR pathway with tissue-specific RNA. The results showed that mrt-2/hus-1/cep-1/ced-4 played a major role in the response phase of radiation side effects. No body was required for signal transduction of radiation damage in the system. Compared with intracellular DDR components, DNA damage checkpoints MRT-2 and HUS-1 in somatic tissues partially contribute to the apoptosis of germ cells induced by inter-system radiation when radiation damage signals are transmitted from the posterior esophageal bulb to the germ cells. Two fluorescent reporting strains CF1553, SJ4100 and molecular probe H2DCF-DA were used to study the ROS expression in nematode whole body after irradiation. The results showed that both of them could induce the increase of ROS level in nematode whole body. Local ROS enhancement was observed in the pharynx and the vagina of the radiation site, and the apoptosis level of the germ cells in the para-region was restored by DMSO treatment, indicating that oxidative damage plays a key role in the transduction of radiation-induced side effects. 5. It was preliminarily proved that ceramide and ceramide-DDR pathway are involved in this process. The deletion of ceramide synthase function can effectively inhibit the apoptosis of germ cells induced by bystander effect, because the apoptosis of germ cells in nematodes is inhibited by either single mutation of lagr-1 (gk327 and hyl-1 (ok976) gene or double mutation of lagr-1 (gk327) and hyl-1 (ok976) gene, while the apoptosis of germ cells in nematodes is inhibited by sphk-1 (0k1097) gene mutation. There was no significant change in the apoptosis of gonad cells, suggesting that ceramide biosynthetic pathway was involved in the induction of radiation-induced bystander effects in nematodes. It has synergistic effect on radiation induced apoptosis of gonadal cells.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q691

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