G-CSF对辐射诱导的造血干细胞损伤的影响及其机制和诱导型一氧化氮合酶对造血系统辐射损伤敏感性的影响

发布时间：2018-05-11 05:25

本文选题：G-CSF + 造血干细胞　；参考：《北京协和医学院》2016年博士论文

【摘要】：随着核电站的建立,人类太空活动的增加,临床上放射诊疗设备的广泛应用,以及核武器恐怖威胁等因素的存在,人类的辐射暴露性可能性日益增加。造血系统对电离辐射(IR)高度敏感,辐射引起的急性骨髓抑制是造成急性放射病中感染、出血、贫血等临床表现的重要病理基础。临床上,可以给予造血细胞因子缓解症状,其中粒细胞集落刺激因子(Granulocyte-colony stimulating factor, G-CSF)是应用最广泛的药物。长期骨髓抑制是辐射远期损伤的主要表现,也是临床进行肿瘤放疗、化疗时最常见的毒副作用之一,直接影响到治疗效果及患者的生存质量。长期骨髓抑制具有迟发性,在临床上容易被忽视,目前尚无有效治疗手段,所以机制研究对长期骨髓抑制的预防和治疗具有重要的意义,是造血干细胞研究关注的热点之一。G-CSF可以促进HSC及粒系前体细胞的增殖和分化,缓解受照后急性骨髓抑制。但是有临床观察显示接受放疗、化疗的患者应用G-CSF后骨髓的恢复能力下降,提示G-CSF可能促进造血干细胞(HSC)的增殖分化但损伤了其自我更新能力。G-CSF对造血干细胞辐射损伤的影响及其机制的研究对G-CSF临床救治骨髓抑制的合理使用具有重要的意义。辐射诱导的组织细胞内自由基水平升高是造血系统损伤的重要分子机制。氧化应激中的活性氧或活性氮可以诱导DNA和脂质损伤,蛋白质氧化等,其中应激反应中产生的NO及过氧亚硝基阴离子(ONOO-)等可以造成多种细胞的病理性损伤。应激产生的NO主要由诱导型NO合酶(Inducible NO Synthase,iNOS)介导。前期研究结果显示,iNOS的特异性抑制剂可以减轻辐射引起的血管内皮损伤。阐明iNOS在辐射导致的组织、细胞损伤中的作用,对辐射损伤的分子机制的研究具有重要的意义。为探讨G-CSF对造血干细胞辐射损伤的影响及其机制,课题第一部分利用4Gy,6Gy亚致死剂量造血系统辐射损伤模型,给予G-CSF治疗检测其对造血干细胞的影响；采用对小鼠受照后30天生存率保护作用最佳给药方案。一个月后检测外周血和骨髓细胞数目,骨髓细胞分型,造血祖细胞集落形成能力(CFU-GM), LSK细胞中活性氧(ROS), p38MAPK和p16表达水平的变化。同时移植骨髓细胞进行连续竞争性移植实验。实验结果显示,G-CSF可以有效缓解辐射引起的外周血白细胞降低和造血祖细胞增殖抑制,G-CSF对受照小鼠的骨髓细胞计数和骨髓细胞分型影响不大,但是却加重了造血干细胞多系再植能力和自我更新能力的损伤；机制研究发现CSF损伤作用主要是进一步提高了辐射诱导的氧化应激通路ROS-p38的表达,造血干细胞的衰老。提示在应用G-CSF救治急性辐射损伤应采取相应的策略防治骨髓功能的远期损伤。为阐明iNOS在辐射导致的组织、细胞损伤中的作用,课题的第二部分利用inos-/-小鼠观察iNOS对辐射诱导的造血系统辐射敏感性和损伤恢复的影响。C57小鼠作为野生对照(WT)。实验结果显示,电离辐射可以诱导inos-/-小鼠骨髓细胞凋亡和造血细胞功能减退。inos-/-小鼠接受6Gy全身照射(TBI)14天后可以引起外周血细胞和骨髓细胞计数降低,HPC, HSC, LSK细胞比例和数目降低,造血祖细胞和造血干细胞功能损伤,破坏造血干细胞稳态。但是,WT小鼠和inos-/-小鼠相比没有显著性差别。表明inos基因缺陷对造血细胞体外照射和小鼠TBI引起的辐射损伤和和辐射损伤恢复没有明显影响。提示可能IR引起氧化应激主要是增加了ROS从而造成DNA损伤和细胞凋亡和衰老等,与炎症通路激活诱导iNOS表达产生大量NO从而造成细胞毒性作用有所区别,也可能是辐射诱导的造血系统多通路损伤作用掩盖了NO的缺失引起的细胞毒性作用。综上所述,根据临床G-CSF应用发现的问题,探讨了G-CSF对造血干细胞功能的影响和其机制,提出G-CSF可以进一步加重辐射诱导的造血干细胞衰老,加重HSC的功能损伤,提示在临床应用G-CSF救治急性辐射损伤的同时,应考虑采取相应的策略防治骨髓功能的远期损伤。为进一步研究造血系统辐射损伤的机制,利用缺陷小鼠,探讨诱导性一氧化氮合酶的作用,为造血系统辐射损伤防护提供研究思路和理论依据。
[Abstract]:With the establishment of nuclear power plants, the increase of human space activities, the widespread use of clinical radiation treatment equipment, and the threat of nuclear weapons terrorism, the exposure possibility of human radiation is increasing. The hematopoietic system is highly sensitive to ionizing radiation (IR), and the acute myelosuppression induced by radiation is the cause of acute radiation sickness. Granulocyte-colony stimulating factor (G-CSF) is the most widely used drug. Long term bone marrow suppression is the main manifestation of long-term radiation injury, and it is also the clinical radiotherapy of tumor. One of the most common toxic and side effects at the time of treatment directly affects the effect of treatment and the quality of life of the patients. Long term bone marrow suppression is delayed, easily neglected in clinical practice, and there is no effective treatment at present. Therefore, the mechanism research is of great significance for the prevention and treatment of long-term bone marrow suppression and is a hot issue in the research of hematopoietic stem cells. Point one.G-CSF can promote the proliferation and differentiation of HSC and granulocyte precursor cells and alleviate acute myelosuppression after exposure. However, clinical observation shows that patients receiving radiotherapy and chemotherapy have decreased the recovery ability of bone marrow after G-CSF, suggesting that G-CSF may promote the proliferation and differentiation of hematopoietic stem cells (HSC) but damage their self-renewal ability.G-CSF The study on the effect of radiation injury on hematopoietic stem cells and its mechanism is of great significance to the rational use of G-CSF in the treatment of bone marrow suppression. The increase of free radical levels in the tissue cells induced by radiation is an important molecular mechanism for the damage of hematopoietic system. Reactive oxygen species or active nitrogen in oxidative stress can induce DNA and lipid damage. The NO and the peroxy nitroso anion (ONOO-) produced in the stress response can cause pathological damage to a variety of cells. The NO produced by stress is mainly mediated by the inducible NO synthase (Inducible NO Synthase, iNOS). The results of the earlier study showed that the specific inhibitors of iNOS could reduce the vascular endothelial damage caused by radiation. To elucidate the role of iNOS in radiation induced tissue and cell damage, it is of great significance to the molecular mechanism of radiation damage. To explore the effect of G-CSF on radiation injury of hematopoietic stem cells and its mechanism, the first part of the subject uses 4Gy, 6Gy sublethal dose of hematopoietic system radiation damage model and G-CSF treatment to detect its hematopoiesis The effect of stem cells; the best regimen of 30 natural survival after exposure to mice. One month later, the number of peripheral blood and bone marrow cells, bone marrow cell typing, hematopoietic progenitor cell colony forming ability (CFU-GM), the changes of reactive oxygen species (ROS), p38MAPK and p16 expression levels in LSK cells. The experimental results showed that G-CSF could effectively alleviate the reduction of leukocyte and the inhibition of hematopoietic progenitor cell proliferation induced by radiation. G-CSF had little effect on the count of bone marrow cells and the type of bone marrow cells in the irradiated mice, but it aggravated the damage of the ability of multiple replanting and self renewal of the hematopoietic stem cells. It is found that the effect of CSF damage is mainly to further enhance the expression of ROS-p38 in the radiation induced oxidative stress pathway and the senescence of hematopoietic stem cells. It suggests that the corresponding strategy should be taken to prevent and cure the long term damage of bone marrow function in the application of G-CSF to treat acute radiation injury. It is the role of iNOS in the tissue and cell damage caused by radiating radiation. The second part of the study used inos-/- mice to observe the effects of iNOS on radiosensitivity and damage recovery of radiation induced hematopoietic system..C57 mice were used as wild control (WT). The experimental results showed that ionizing radiation could induce apoptosis of bone marrow cells in inos-/- mice and hematopoietic cell dysfunction in.Inos-/- mice to receive 6Gy whole body irradiation (TBI) for 14 days. After that, the number of peripheral blood cells and bone marrow cells decreased, the proportion and number of HPC, HSC, LSK cells decreased, the function of hematopoietic progenitor cells and hematopoietic stem cells were damaged and the hematopoietic stem cell homeostasis was destroyed. However, there was no significant difference between the WT mice and the inos-/- mice. It showed that the iNOS gene defects were induced in vitro of hematopoietic cells and induced by TBI in mice. It is suggested that the oxidative stress caused by IR may cause DNA damage and cell apoptosis and senescence mainly caused by ROS induced oxidative stress, which can lead to a large number of NO resulting from the activation of the inflammatory pathway to induce the expression of iNOS, and may also be a multipass hematopoietic system induced by radiation. The effect of road damage conceals the cytotoxic effect of NO loss. To sum up, according to the problems found in the clinical G-CSF application, the effect of G-CSF on the function of hematopoietic stem cells and its mechanism are discussed. It is suggested that G-CSF can further aggravate the senescence of hematopoietic stem cells induced by radiation and the function damage of heavy HSC, suggesting that G-CSF can be used in clinical application. In order to cure the acute radiation injury, we should consider the corresponding strategy to prevent the long-term damage of the bone marrow function. In order to further study the mechanism of the radiation damage of the hematopoietic system, the effect of inducible nitric oxide synthase (inducible nitric oxide synthase) is discussed by using the defective mice, and the research ideas and theoretical basis for the protection of the radiation injury of the hematopoietic system are provided.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R818

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