【作者】 雷润宏；

【导师】 邓玉林；

【作者基本信息】 北京理工大学 ， 生物化工， 2015， 博士

【摘要】 随着载人空间站作为国家重大科技专项被批准,载人登月和火星探测等国家重大科技专项也进入了论证或等待批准阶段。这些国家重大科技工程的论证和批准,既为空间生命科学研究提供了前所未有的机遇,也提出了长期载人航天飞行的安全性问题。从近地轨道向其他星体的不断探索,使得人类在空间特殊环境中暴露的时间超过六个月。这种长期暴露对宇航员自身造成的影响,是由空间环境多种因素(辐射、微重力、狭小空间等)联合作用造成的,然而电离辐射,特别是重离子辐射,因其具有累积生物效应,造成在六个月以上的飞行期间,宇航员大约三分之一的DNA将直接被电离辐射击中;此外,重离子辐射不仅能直接造成靶器官的损伤,而且还能引发旁效应,对非靶器官造成影响。而当前关于空间重离子辐射生物学效应的研究,主要以相对较低剂量(<4 Gy)、全身辐射、短期(<7天)和重叠效应为主要特点,造成在多器官损伤情况下,难以对辐射损伤的级联反应调控及响应次序进行阐释。因此,研究局部器官辐射损伤,特别是脑局部空间重离子辐射损伤后的直接效应及其旁效应,有助于明确空间重离子全身辐射损伤的潜在机制,进而为辐射防护策略的制定提供依据。针对空间重离子辐射研究中对长期效应,包括中枢神经系统损伤及其旁效应研究的不足,本课题从如下五个方面进行了研究。第一,以12C6+为离子源,建立了脑局部重离子辐射损伤动物模型,并对中枢神经系统的长时程(辐射后1个月,2个月,3个月)损伤效应进行评估。第二,对中枢神经系统辐射损伤引起的胸腺、脾脏和外周血中的长时程旁效应进行了系统研究。第三,全面评估了中枢神经系统损伤引起的在其他外周器官,包括心肌、肺脏、肝脏、气管、肾脏、胃和主动脉中的长时程旁效应。第四,在细胞模型上研究了重离子辐射后神经元和神经胶质细胞对免疫细胞旁效应的介导作用。第五,在细胞模型上研究了神经胶质细胞在重离子辐射条件下辐射耐受性的机制。通过上述研究,获得如下结果。首先建立了脑局部重离子辐射损伤的动物模型,并发现中枢神经系统出现神经元萎缩及细胞凋亡。选用Wistar大鼠(体重180 g±10g),在中国科学院近代物理研究所兰州重离子加速器平台癌症治疗终端进行辐射暴露;选用高能12C6+离子束为离子源,能量为165 MeV/u,传能线密度为30 KeV/μm,辐射剂量率为0.3~0.5 Gy/min,坪区辐射吸收总剂量为15 Gy;大鼠脑的射线暴露区域为眼球后缘至枕骨前缘,射线以垂直方式进入暴露区域。脑局部重离子辐射对中枢神经系统的长时程效应中,病理检测发现区域性神经元萎缩,主要在顶叶皮层和枕叶皮层;全脑tunel染色发现,从辐射后第一个月到第三个月,均有广泛分布的特征性凋亡细胞。第二,脑局部重离子辐射使大鼠外周免疫器官(胸腺、脾脏)及外周血细胞发生显著改变。首先对于胸腺,与对照组相比,萎缩进程加快,胸腺皮质变薄,tunel阳性胸腺细胞数量明显增多,且随辐射后时间延长,数量增加;胸腺内氧化应激水平发生紊乱;t细胞分化异常,相关基因c-kit、rag1、sca1等在转录水平上发生下调,而cd8+t细胞比例明显增加;胸腺微环境发生改变,表现为糖皮质激素(gc)及其受体(gr)依赖的细胞凋亡通路的激活,以及胸腺分泌炎症因子的功能紊乱;胸腺中cd3+t淋巴细胞在辐射后前两个月并未发生明显减少;转录组分析发现,与细胞粘附相关的多条通路均发生改变,导致胸腺自身病原清除能力下降。对于脾脏,脑局部重离子辐射引起含铁血红素吞噬类细胞数量明显增加(约10倍);在辐射后第三个月,脾脏基质细胞大幅减少(46.7%);tunel阳性脾脏细胞随着辐射后时间的延长,细胞数量明显增加(4~7倍);脾脏中t细胞分布紊乱,表现为辐射后第一个月和第二个月cd3+cd4–cd8+t淋巴细胞增加,而第二个月和第三个月cd3+cd4+cd8–t淋巴细胞比例增加。进一步研究发现,脾脏分泌tnf-α、ifn-γ、il-6和ssao的功能发生紊乱,免疫抑制标志分子il-10在第二个月和第三个月明显增加,提示脾脏发生免疫抑制。此外,与对照组相比,外周血中白细胞在辐射后第一个月增加21.4%,在辐射后第二个月和第三个月则分别减少26.4%和33.3%。总淋巴细胞数在辐射后第二个月和第三个月明显下降,与各自对照组比,减少28.6%和43.6%,提示免疫功能受损,而t淋巴细胞总数相对恒定;红细胞在辐射后第三个月减少8.9%,而血小板在辐射后第一个月增加33.6%。第三,脑局部重离子辐射引起其他外周器官不同程度的损伤。大鼠15gy脑局部重离子辐射引起心肌萎缩、局灶性纤维化,促炎因子和炎症因子(ssao,peg2,inos,il-6,tnf-α和ccl20)表达增加;肺脏血管周围水肿、tunel阳性细胞数量增加;肝脏tunel阳性细胞数量增加;辐射后第一个月,气管上皮凋亡细胞数量明显增加,而辐射后第三个月,气管粘膜和粘膜下层凋亡细胞数量增加;肾脏在辐射后第一个月没有tunel阳性细胞,而在第三个月时凋亡细胞数量明显增加;食管tunel阳性细胞数量明显增加,胃部粘膜上皮细胞凋亡增加,并出现萎缩;主动脉中ssao,tnf-α,ccl20,inos,il-6和pge2的蛋白含量均在辐射后第三个月明显升高,caspase-3蛋白含量也明显增加。总之,脑局部重离子辐射引发广泛的旁效应,全身重要的脏器均出现不同程度的损伤,并且总体上实质性器官比非实质性器官损伤严重。第四,条件性培养基中的细胞因子能够介导免疫细胞的旁效应。基于共培养的细胞模型研究发现,单独神经胶质细胞(u87)的条件性培养基能够促进thp-1和jurkat细胞活力增加,而神经元(sh-sy5y)的条件性培养基对免疫细胞的促进作用并不明显,u87的条件性培养基对不同免疫细胞的活化程度,从高到低依次为thp-1,u937和jurkat;通过细胞因子微球检测技术(cytometricbeadarray,cba)发现,条件性培养基中il-2,il-6,il-10,il-12,mcp-1,mig,mip-1α,mip-1β含量的变化幅度与种类丰度,与免疫细胞的活化程度密切相关。神经胶质细胞和神经元共培养之后的条件性培养基能够显著增加u937的细胞活力,其与tnf-α,mig和il-12含量的增加有关。进一步的探索发现,共培养神经胶质细胞和神经元与两种细胞单独培养所产生的条件性培养基对不同免疫细胞的活化的差异,与神经元和神经胶质细胞之间膜结构依赖的物质交换相关,即二者能够通过囊泡进行非接触式的信息交换,如sh-sy5y能够摄取u87的细胞膜,而u87能够将sh内表达的绿色荧光蛋白摄取到其细胞核周围。在重离子辐射条件下,神经细胞的条件性培养基,能够对不同的免疫细胞产生不同的效果,这种变化与辐射剂量相关。通过检测发现,培养基中因辐射导致的细胞因子的变化,包括il-2,il-10,il-12/il-23,mig,mip-1α和mip-1β,是导致不同免疫细胞响应不同的直接原因。特别地,重离子辐射共培养神经细胞来源的条件性培养基,能够显著性抑制thp-1细胞的分化和迁移,达到52.6%。提示,细胞因子可能是神经细胞辐射引发免疫系统旁效应的媒介分子。第五,建立了重离子辐射损伤的细胞模型,发现核内调控因子hmgb1在胞内通过引发自噬,增加其重离子辐射耐受性。本研究选用神经胶质瘤细胞u251,以能量为80.55mev/u,let为75kev/μm,辐射剂量率为3gy/min的12c6+离子束辐射,辐射吸收剂量梯度为1gy,2gy,5gy,辐射后24h,48h,72h收集细胞和培养基,进行检测。结果表明,通过丙酮酸乙酯(ethylpyruvate)和pyridone6抑制hmgb1向细胞外的释放,使其在胞内含量增加,将导致u251细胞辐射耐受性增强,表现为存活率升高;低剂量(1gy)重离子辐射及辐射后24h,细胞内hmgb1水平显著性升高,此时,自噬水平(lc3b、beclin1表达)也显著性升高;在5gy剂量辐射及辐射后72h,细胞内hmgb1水平逐渐降低,而胞外含量增加,同时lc3b和beclin1胞内含量也明显降低,凋亡水平明显升高(caspase3/7、caspase-8活性增加,抗凋亡蛋白c-FLIP水平降低,凋亡执行蛋白caspase-3的前体蛋白减少)。提示,在一定剂量(1 Gy)和时间(24 h)内,HMGB1在细胞内发挥促进自噬发生的作用,从而增加其辐射耐受性。但是随着辐射剂量增加或者辐射后时间延长,辐射引起的凋亡使得HMGB1释放到细胞外,胞内含量减少,进而使自噬水平降低,细胞出现死亡。提示,以HMGB1相关的自噬通路作为靶点,进行药物干预,能够对临床以重离子为工具进行放化疗联合治疗神经胶质瘤提供新的策略。开展模拟长期载人航天飞行条件下重离子辐射对神经系统损伤及其旁效应的研究,有助于阐明空间重离子全身辐射后机体损伤重叠效应的级联次序,从而加深对空间辐射损伤的认识、评价和预防,可以为空间辐射损伤生物标志预警体系提供理论依据,对于我国载人空间站、载人登月以及火星探测等国家重大科技工程,具有很好的理论意义和应用前景。另外,近年来肿瘤的重离子治疗已经受到广泛关注并成为肿瘤放疗的新技术而得到应用。重离子辐射损伤及其耐受性的深入研究也将有助于加深对重离子治疗新技术的认识,从而推动其广泛的临床应用。更多还原

【Abstract】 The set up of China manned space station promoted the proofing of the key state science and technology projects. China could make the first manned moon landing and send a probe to Mars after full discussion and demonstration. These efforts not only raise a security issue in the long-term manned space flight, but also provide opportunities for space life science research. During the journey of exploration from the Earth to other planets, people will be exposed to outer space environment more than six months, which inflicts damage on DNA to the amount of one third in the body. Even more, bystander effects can be induced in non-targeted organs. However, current studies related to biological effects of space heavy ion radiation mainly focus on the relative low dose(< 4 Gy), whole body irradiation, short term(< 7 days) and superimposed effects, that leads to the importance of explaining the cascade reactions and regulatory sequences after irradiation. Therefore, investigation about the direct and indirect effects after localized irradiation, especially the brain localized heavy ion irradiation would greatly benefit the understanding about the mechanism of damage after whole body space heavy ion irradiation and the development of astronaut heath care technology.Due to the deficiencies in the long-term effects of space heavy ion radiation and the brain localized radiation induced bystander effects, this study was conducted and focused on some points as follows: Establishment of a rat model with brain localized heavy ion irradiation and investigation of the direct effects in center nervous system one, two and three months after irradiation; profound study about the bystander effects in thymus, peripheral blood and spleen; evaluation of the bystander effects in peripheral organs, including cardiac muscle, lung, liver, trachea, kidney, stomach and aorta; finding of conditioned medium mediated effects from neuron and glia cells to immune cells; explanation of the heavy ions irradiation tolerance of glioma cells.Based on the design above, some novel findings were observed. Firstly, we established a rat model with brain localized heavy ion irradiation. Wistar rats, with the body weight of 180 g ± 10 g were exposed at Heavy Ion Research Facility in Lanzhou(HIRFL). Rats in the experimental group were irradiated with single high dose of 15 Gy vertically on the back of the head with a 12C6+ ion beam(primary energy, 165 MeV/u; LET, 30 KeV/μm; intensity, 0.3~0.5 Gy/min). Long-term biological effects on the central nervous system, including neuronal atrophy and cell apoptosis were examined. The data showed that atrophy in the parietal cortex and occipital cortex was induced. Widely scattered TUNEL-positive cells were found from one to three months after irradiation, which indicates the presence of extensive injury of the rat brain subjuected to direct irradiation.Secondly, we evaluated the local brain irradiation derived damages in the peripheral immune organs(thymus and spleen) and blood. In the thymus, atrophy process was accelerated after brain-localized heavy ion radiation. The cortex thinning and increased number of apoptosis cells were found, which presented time-dependent features. Compared with the thymus in control group, oxidative stress and T-cell development were disturbed. Genes related to thymic T-cell development, such as c-kit, Rag1 and Sca1 were down-regulated in transcriptional level. However, the proportion of CD8+ T-cells increased significantly. Thymic microenvironment was also affected. Glucocorticoid and its receptor related apoptotic pathway was triggered and inflammatory factors secretion was disordered. Whereas, CD3+ T-cells were not apparently decreased. Transcriptome sequencing showed that multiple pathways associated with cell adhesion were changed, which led to the decrement of thymus pathogen clearance ability. In the spleen, number of stromal cells decreased significantly while the number of hemosiderin-positive macrophages, extracellular matrix, and TUNEL-positive cells increased compared with that in control group. T cells subtypes distribution was also disturbed. The proportion of CD3+CD4–CD8+ and CD3+CD4+CD8– T-cells elevated in chronological order. In addition, inflammation and immunosuppression related secretion, including TNF-α, IFN-γ, IL-6, SSAO and IL-10 were triggered. In the peripheral blood, the number of total lymphocyte and leukocyte decreased notably. Red blood cells and platelet were also affected. However, the total number of T lymphocyte did not altered.Thirdly, brain-localized heavy ion radiation could induce considerable injury in peripheral organs. Increased myocardial hypotrophy, focal fibrosis and inflammation factors(SSAO, PEG2, i NOS, IL-6, TNF-α and CCL20) were found. In lungs, edema surrounding blood vessels and apoptosis were observed. Epithelial cells in the trachea presented apoptosis one month after irradiation while mucosa and submucosa presented apoptosis three months after irradiation. In kidney, apoptosis was found only at three months after irradiation. Gastric muosa epithelial cells showed great TUNEL-positive signal and atrophy after irradiation. SSAO, TNF-α, CCL20, iNOS, IL-6 and PGE2 increased significantly three months after irradiation. Hence, evident bystander effects in fundamental peripheral organs were caused after brain localized heavy ion irradiation. In general, parenchymatous organs showed great injury than that in non-parenchymatous organs.Fourthly, cytokines in conditioned medium could mediate bystander effect on immune cells. Based on a neuro-immune interaction cell model, we found that U87 conditioned medium can strongly enhance THP-1 and Jurkat cell viability and the SH or U87 and SH co-culture conditioned medium made weak effect compared to the U87 conditioned medium. But for U937 cells, the viability was significantly enhanced after U87 and SH conditioned medium treatment, which was even higher than that treated with U87 conditioned medium. U87 conditioned medium can evidently enhance the cell viability of THP-1, U937 and Jurkat cells. Presumably, synergistic effect of the cytokines may be involved in the activations. Then, cytokines and chemokines involved in the interaction and their effects were studied. Certainly, different combination of glioblastoma cell and neuroblastoma cell resulted in different secretions, which lead to different effect on different immune cells. This observation implicated that direct communication would be progressed in the co-culture condition. Information exchange could occur via liposome, exosome, as well as other mediators which contain various molecular constituents, including proteins and RNA. Membrane vesicle trafficking was found in SH or U87 and SH co-culture conditions. Effects of the neural cells damage after irradiation on peripheral immune cells were evaluated. Medium conditioned by neural cells receiving 5 Gy of heavy ion radiation increased the viability of both THP-1 and Jurkat cells compared with that of medium conditioned by mock-irradiated neural cells. A significant reduction of migrated THP-1 cells was caused after exposure to the conditioned medium of irradiated cells. These data indicate that neural cell injury caused by carbon ion radiation may enhance the proliferation of peripheral immune T-cells and decrease the migration and invasion ability of monocytes.Fifthly, based on a cell model of heavy ion radiation, HMGB1 mediated regulation of U251 cells heavy ion radiation tolerance was found. Glial cell could play its role in the form of independent neurons and is of great importance in supporting neurons in structure and function to maintain the activity of central nervous system. After exposure to 12C6+ ion beam(80.55 MeV/u primary energy; LET, 75 KeV/μm; intensity, 3 Gy/min), the data demonstrated that, heavy ion radiation could greatly reduce cell survival in a time- and dose-dependent manner. Morphological changes, such as the enlarged cell size and tenuous shape were also observed. Meanwhile, autophagy was efficiently induced, which then decreased in a time- and dose-dependent manner. HMGB1 expression in the cells presented important correlation to changes of autophagy. Pharmacological inhibition of HMGB1 release by ethyl pyruvate and pyridone 6 significantly decreased heavy ion radiation induced glioblastoma cell death. These results further conformed the important role of HMGB1 in radio-resistance of glioma cells.Research about the injury in central nervous system and its bystander effects under simulated space heavy ion radiation condition in the manned space flight is conducive to deeply understand the space radiation risk and injury-relevant evaluation and prevention, as well as further clarification of whole-body irradiation induced overlapping effect. Theoretical foundation can be supplied to the early warning system in space radiation. Besides, it also has theoretical value and application prospect to the key state science and technology projects, such as the establishment of manned orbital station, human lunar exploration and Mars probes launch. Moreover, cancer radiotherapy by using heavy ions has been greatly used. Intensive studies about the heavy ion radiobiological effects could be beneficial to the heavy ions based radiotherapy technique, which thereby promotes its clinical application.更多还原