照射后造血干细胞促进辐射损伤小鼠造血及免疫功能修复的实验室研究

发布时间：2018-06-21 03:45

本文选题：急性辐射损伤综合症 + 异基因造血干细胞　；参考：《中国人民解放军军事医学科学院》2011年博士论文

【摘要】：辐射损伤后造血及免疫功能恢复迟缓是导致ARS患者死亡的主要原因。辐射剂量越大,造血及免疫功能受损越严重。根据欧洲血液与移植合作组于2005年推出的核事故医学救治指南(METREPOL)的分级标准,将血液(H)、消化道(G)、神经系统(N)、及皮肤(C)损伤分为1-4级,分级越高,损伤越严重。1级为组织器官功能能够完全恢复正常,而4级则为组织器官致命的、不可逆损伤,无自身恢复的可能性,2、3级介于两者之间。分级不同,患者救治的原则不同。仅造血系统而言,H1级患者可暂不作处理,密切观察、随访,H2-4级患者由于造血受到不同程度的抑制,需要给与积极有效的治疗。造血干细胞移植虽然能够快速恢复患者造血,但它首先受到供者来源的限制,辐射损伤后短时间内很难找到HLA配型全相合的异基因造血干细胞供者,而且异基因造血干细胞移植后GVHD、免疫重建困难等并发症同样是导致ARS患者死亡的重要原因,因此,METREPOL推荐异基因造血干细胞移植仅适用于不合并严重烧伤及其他组织创伤的H4级患者。而H2-3级患者主要依靠细胞因子、成份血制品输注等对症支持治疗。但H3级以及部分介于H3-H4级之间的患者,由于自身残存造血干细胞量少,在常规支持治疗下血象恢复缓慢,导致此部分患者在相当长的时间内面临出血、感染而继发死亡的风险。因此如何促进此部分患者的造血及免疫恢复,减少相关并发症、提高其生存是本课题研究的重点。很多研究尝试利用造血干细胞能够在体内快速倍增的特点,发挥其支持造血的作用,但自体造血干细胞由于辐射损伤后残存数量较少,很难有实际应用价值,异基因造血干细胞虽然数量上有保证,但由于辐射损伤后患者免疫功能低下,异基因造血干细胞很容易发生稳定高比例植入,取代患者自身造血,从而导致GVHD等并发症发生。从既往ARS患者治疗过程中发现,造血干细胞一过性植入,同样能够起到支持造血的作用,而GVHD的发生与供者造血干细胞在受者体内的植入比例密切相关,如何通体外处理降低供者造血干细胞的生长、增殖活性,使其仅在受者体内短时间、低比例植入,起到促进受者自身造血恢复,同时避免GVHD的发生是本课题重点要解决的问题。与其他途经相比,体外照射是降低造血干细胞活性最为简单有效的方法,在第一部分的研究中我们,利用半相合小鼠供受者模型(CB6F1→BALB/c),将动员后供者脾脏造血干细胞分别给与2.5Gy、5Gy、7.5Gy、10Gyγ射线照射后输入6 Gy亚致死剂量辐射损伤的受者小鼠体内(3x107/只),发现照射后供者造血干细胞能够加快受者小鼠的造血恢复,而且体外照射剂量越小,支持造血的作用越明显。以2.5Gy照射细胞效果最好,5Gy、7.5Gy照射细胞次之,10Gy照射细胞对造血几乎无明显促进作用。同时我们利用流式细胞仪联合实时定量PCR的方法检测供者细胞在受者小鼠体内的植入情况发现,2.5Gy照射供者造血干细胞在部分受者小鼠外周血中形成稳定、高比例植入(95%),从而取代受者造血,引发GVHD导致小鼠死亡。而5Gy照射造血干细胞且仅在受者外周血中形成微量植入/嵌合(10-3-10-4水平),持续2-4月后被受者完全被排掉,观察至术后半年,无一例小鼠发生GVHD。照后1月,未在受者外周血中检测到经7.5Gy、10Gy照射的供者细胞成分,两组小鼠同样无GVHD发生。通过上述实验我们发现7.5Gy、5Gy照射供者造血干细胞输注能够起到既支持受者自身造血恢复又无GVHD发生的作用,为ARS的临床治疗提供了新思路。随后我们将7.5Gy、5Gy照射供者造血干细胞与G-CSF联合后观察其对6 Gy亚致死剂量以及8Gy致死剂量辐射损伤小鼠的影响,发现联合后对造血恢复有很好的协同作用,尤其是能够显著提高血红蛋白及血小板的水平,而且能大幅提高8Gy辐射损伤小鼠生存,其中以5Gy照射细胞联合G-CSF效果最为明显,显著优于单独使用G-CSF(70+10.1% VS 30+10.1%)。植入检测发现,7.5Gy照射细胞在8Gy辐射损伤小鼠体内形成微量嵌合,2-4个月内被受者排掉。5Gy照射细胞在8Gy辐射损伤小鼠体内的植入比例差别较大,从微量嵌合到高比例植入,但均不稳定,2-4个月内被排掉,均恢复受者小鼠自身造血,两细胞治疗组无一例发生GVHD。这对于重度骨髓型放射病有极少量残存自身造血干细胞患者的临床治疗有重要参考价值。我们继续将上述剂量体外照射的造血干细胞与G-CSF联合用于超大致死量10Gy辐射损伤小鼠的救治。结果发现,7.5Gy照射造血干细胞+G-CSF组、G-CSF组以及对照组小鼠全部死亡,而5Gy照射造血干细胞+G-CSF组小鼠存活率达到60%,造血快速恢复,但5Gy照射供者造血干细胞在受者小鼠体内形成高比例稳定嵌合,快速的造血恢复来源于供者细胞的完全植入取代受者造血,而并非受者自身造血恢复,因此导致部分小鼠发生慢性GVHD死亡,提示在受者小鼠无残存自身造血的情况下,体外照射的造血干细胞治疗可能存在的风险。在第一部分中我们还就照射后造血干细胞体外增殖能力、体内归巢以及其支持造血的机理进行了初步研究,并在正文中对造血干细胞体外灭活程度、受者免疫抑制程度、供者细胞植入比例与GVHD发生关系进行了详细分析。在第二部分的研究中,我们选取在第一部分中能够起到良好造血促进作用的5Gy照射供者造血干细胞与G-CSF联合方案,观察其对6Gy亚致死量辐射损伤小鼠免疫功能的影响。结果提示联合治疗能够调整辐射损伤小鼠中枢及外周淋巴细胞亚群的比例,促进淋巴细胞功能恢复。G-CSF联合细胞输注后可促进胸腺双阴性细胞的增殖、分化,减轻双阳性细胞凋亡,促进其向CD4、CD8单阳性细胞分化,提高外周血淋巴细胞绝对值,以及CD3+CD4+、CD3+CD8+细胞比例,增强T、B淋巴细胞对丝裂原的反应。利用实时定量PCR对胸腺TRECs水平检测我们发现照后30d、60d,G-CSF联合细胞输注组TRECs拷贝数明显高于对照组及单独使用G-CSF组,流式细胞仪检测受者小鼠外周初始T细胞水平发现联合治疗组外周血中初始T细胞水平明显增高,提示联合治疗可增加胸腺近期输出功能,促进辐射损伤后中枢免疫功能重建。通过上述实验研究,我们发现G-CSF联合体外一定剂量照射后半相合异基因造血干细胞输注能够促进亚致死量及有极少残存造血的致死量辐射损伤小鼠自身造血及免疫功能恢复,无GVHD等副反应发生,为ARS的细胞治疗提供了一条新思路、新方法。
[Abstract]:Hematopoiesis and retardation of immune function recovery after radiation injury are the main causes of death in ARS patients. The greater the radiation dose, the more serious the impairment of hematopoiesis and immune function. According to the classification criteria of the medical treatment guide for nuclear accidents (METREPOL), launched in 2005 by the European Cooperation Group on blood and transplantation, the blood (H), the digestive tract (G), and the nervous system (N) are used, and The skin (C) damage is divided into 1-4 grades, the higher the classification, the more serious the damage to the.1 level, the organ function can be completely restored, and the 4 level is the fatal, irreversible injury, no self recovery possibility, and the 2,3 level is between the two. The classification is different, the treatment principle of the patients is different. Only the hematopoietic system, the H1 patients can not do it temporarily. Treatment, close observation, follow-up, H2-4 patients need to be given active and effective treatment because of different levels of hematopoiesis. Hematopoietic stem cell transplantation can quickly restore the patient's hematopoiesis, but it is first restricted by donor sources. It is difficult to find a HLA matching allogenic hematopoietic stem cell for a short time after radiation injury. GVHD, the difficulty of immune reconstruction and other complications after allogeneic hematopoietic stem cell transplantation is also an important cause of death in ARS patients. Therefore, METREPOL recommends allogeneic hematopoietic stem cell transplantation only for patients with H4 level without severe burns and other tissue injuries. And H2-3 patients rely mainly on cytokine, component blood system. However, patients with H3 and part of the H3-H4 level, which have less hematopoietic stem cells in their own remnants, have a slow recovery of hemogram under conventional support, leading to the risk of bleeding, infection and secondary death in this part of the patient for a long time. Therefore, how to promote hematopoiesis and the hematopoiesis in this part of the patient. It is the focus of our research that immune recovery, reduction of related complications and improvement of their survival.
Many studies try to make use of the characteristics of hematopoietic stem cells which can rapidly multiplier in the body and play its role in supporting hematopoiesis. However, the number of autologous hematopoietic stem cells is very difficult to be applied in the actual application because of the small number of remnants after radiation damage. Although the number of allogeneic hematopoietic stem cells is guaranteed, the immune function of the patients is low after radiation injury, Allogeneic hematopoietic stem cells are prone to stable high proportion implantation and replace the patient's own hematopoiesis, which leads to the occurrence of GVHD and other complications. From the previous treatment of ARS patients, it was found that the hematopoietic stem cell implants can also play a role in supporting hematopoiesis, and the ratio of GVHD to donor hematopoietic stem cells in the recipient's body Cases are closely related, how the treatment reduced cell growth, donor hematopoietic stem cell proliferation, the recipient of short time, low proportion of implants, to promote their own hematopoietic recovery, while avoiding the occurrence of GVHD is the key to solve the problem.
In comparison with other approaches, in vitro irradiation is the most simple and effective way to reduce the activity of hematopoietic stem cells. In the first part of the study, we used the semi matched mouse donor model (CB6F1 to BALB/c) to give 2.5Gy, 5Gy, 7.5Gy, and 10Gy gamma radiation into the 6 Gy sublethal dose radiation of the donor spleen hematopoietic stem cells respectively. In the injured recipient mice (3x107/ only), it was found that the donor hematopoietic stem cells could accelerate the recovery of hematopoiesis in the recipient mice after irradiation, and the smaller the irradiation dose, the more obvious the effect of supporting hematopoiesis. The best effect of 2.5Gy irradiated cells, 5Gy, 7.5Gy irradiated cells, and 10Gy irradiated cells had little effect on hematopoiesis. We detected the implantation of donor cells in the recipient mice by flow cytometry combined with real-time quantitative PCR. It was found that the 2.5Gy irradiated donor stem cells were stable in the peripheral blood of some of the recipient mice, with a high proportion of implantation (95%), thus replacing the recipient hematopoiesis and causing GVHD to lead to the death of mice. And 5Gy irradiated the hematopoietic stem cells. And only in the peripheral blood of the recipient, microimplantation / chimerism (10-3-10-4 level) was formed, and the recipient was completely removed after 2-4 months. After six months of the operation, no mice had GVHD. illumination in January, and the donor cells irradiated by 7.5Gy and 10Gy were not detected in the peripheral blood of the recipient, and the two groups of mice had no GVHD. It is found that 7.5Gy, 5Gy irradiated donor hematopoietic stem cell infusion can not only support the recovery of the recipient's own hematopoiesis but also have no GVHD, which provides a new idea for the clinical treatment of ARS. Then, we combined 7.5Gy, 5Gy irradiated donor stem cells and G-CSF to observe the 6 Gy sublethal dose and 8Gy lethal dose radiation injury mice. It is found that the combined effect of combination on hematopoiesis has a good synergy, especially the level of hemoglobin and platelets, and can significantly improve the survival of 8Gy irradiated mice, in which the effect of 5Gy irradiated cells combined with G-CSF is most obvious, which is significantly better than single single G-CSF (70+10.1% VS 30+10.1%). 7.5Gy irradiated cells formed microchimerism in mice with 8Gy radiation injury, and the proportion of the implanted.5Gy irradiated cells in the 8Gy radiation injured mice within 2-4 months was different, from microchimerism to high proportion implantation, but all were unstable and were discharged within 2-4 months, all of them recovered to the recipient mice's own hematopoiesis, and no one in the two cell treatment group. GVHD. has an important reference value for the clinical treatment of severe myeloid radiation disease with a very small amount of remnants of their own hematopoietic stem cells. We continue to combine the above dose of hematopoietic stem cells with G-CSF in vitro for the treatment of 10Gy irradiated mice with ultra large lethal dose. The results showed that 7.5Gy irradiation of hematopoietic stem cells was +G-CSF. All the mice in the group, G-CSF group and the control group died, while the survival rate of 5Gy irradiated hematopoietic stem cells in the +G-CSF group was 60% and the hematopoiesis was recovered quickly, but the 5Gy irradiated donor stem cells formed a high proportion of stable chimerism in the recipient mice, and the rapid hematopoiesis came from the complete implantation of donor cells to replace the recipient hematopoiesis, but not the recipient. The recovery of autologous hematopoiesis leads to chronic GVHD death in some mice, which suggests the possible risk of hematopoietic stem cell therapy in vitro. In the first part, the proliferation of hematopoietic stem cells after irradiation, homing in the body, and the mechanism of its support for hematopoiesis are also introduced in the first part. The preliminary research, and in the body of hematopoietic stem cells in vitro inactivation, the recipient immune suppression and donor cells into the relationship between proportion and the GVHD occurrence are analyzed in detail.
In the second part of the study, we selected the combined scheme of 5Gy irradiated donor hematopoietic stem cells and G-CSF in the first part to observe its effect on the immune function of mice induced by 6Gy sublethal radiation. The results suggest that combined therapy can adjust the central and peripheral lymphocyte subsets of radiation injured mice. The proportion of the group, promoting lymphocyte function recovery after.G-CSF combined cell infusion can promote the proliferation, differentiation, and decrease the apoptosis of double positive cells, promote its differentiation to CD4, CD8 single positive cells, increase the absolute value of peripheral blood lymphocytes, and the proportion of CD3+CD4+, CD3+CD8+ cells, T, B lymphocyte against mitogen. According to the detection of thymus TRECs level by real-time quantitative PCR, we found that the number of TRECs copies in 30d, 60d, G-CSF combined cell infusion group was significantly higher than that of the control group and the single G-CSF group. The level of initial T cells in the peripheral blood of the combined treatment group was significantly higher than that of the G-CSF group, and the flow cytometer detected the initial T cell level in the peripheral blood of the combined treatment group, suggesting the combination of the initial T cell level in the peripheral blood of the combined treatment group. Treatment can increase the recent output function of thymus and promote the reconstruction of central immune function after radiation injury.
Through these experimental studies, we found that G-CSF combined with a certain dose of allogeneic hematopoietic stem cell infusion after a certain dose of irradiation in vitro can promote the sublethal dose and the recovery of the hematopoiesis and immune function of mice with minimal residual hematopoiesis, without the occurrence of GVHD and other side reactions, which provides a new way of thinking for the cell therapy of ARS. New method.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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