本文選題：急性輻射損傷綜合癥 + 異基因造血干細(xì)胞��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年博士論文

【摘要】：輻射損傷后造血及免疫功能恢復(fù)遲緩是導(dǎo)致ARS患者死亡的主要原因。輻射劑量越大,造血及免疫功能受損越嚴(yán)重。根據(jù)歐洲血液與移植合作組于2005年推出的核事故醫(yī)學(xué)救治指南(METREPOL)的分級(jí)標(biāo)準(zhǔn),將血液(H)、消化道(G)、神經(jīng)系統(tǒng)(N)、及皮膚(C)損傷分為1-4級(jí),分級(jí)越高,損傷越嚴(yán)重。1級(jí)為組織器官功能能夠完全恢復(fù)正常,而4級(jí)則為組織器官致命的、不可逆損傷,無(wú)自身恢復(fù)的可能性,2、3級(jí)介于兩者之間。分級(jí)不同,患者救治的原則不同。僅造血系統(tǒng)而言,H1級(jí)患者可暫不作處理,密切觀察、隨訪,H2-4級(jí)患者由于造血受到不同程度的抑制,需要給與積極有效的治療。造血干細(xì)胞移植雖然能夠快速恢復(fù)患者造血,但它首先受到供者來(lái)源的限制,輻射損傷后短時(shí)間內(nèi)很難找到HLA配型全相合的異基因造血干細(xì)胞供者,而且異基因造血干細(xì)胞移植后GVHD、免疫重建困難等并發(fā)癥同樣是導(dǎo)致ARS患者死亡的重要原因,因此,METREPOL推薦異基因造血干細(xì)胞移植僅適用于不合并嚴(yán)重?zé)齻捌渌M織創(chuàng)傷的H4級(jí)患者。而H2-3級(jí)患者主要依靠細(xì)胞因子、成份血制品輸注等對(duì)癥支持治療。但H3級(jí)以及部分介于H3-H4級(jí)之間的患者,由于自身殘存造血干細(xì)胞量少,在常規(guī)支持治療下血象恢復(fù)緩慢,導(dǎo)致此部分患者在相當(dāng)長(zhǎng)的時(shí)間內(nèi)面臨出血、感染而繼發(fā)死亡的風(fēng)險(xiǎn)。因此如何促進(jìn)此部分患者的造血及免疫恢復(fù),減少相關(guān)并發(fā)癥、提高其生存是本課題研究的重點(diǎn)。 很多研究嘗試?yán)迷煅杉?xì)胞能夠在體內(nèi)快速倍增的特點(diǎn),發(fā)揮其支持造血的作用,但自體造血干細(xì)胞由于輻射損傷后殘存數(shù)量較少,很難有實(shí)際應(yīng)用價(jià)值,異基因造血干細(xì)胞雖然數(shù)量上有保證,但由于輻射損傷后患者免疫功能低下,異基因造血干細(xì)胞很容易發(fā)生穩(wěn)定高比例植入,取代患者自身造血,從而導(dǎo)致GVHD等并發(fā)癥發(fā)生。從既往ARS患者治療過(guò)程中發(fā)現(xiàn),造血干細(xì)胞一過(guò)性植入,同樣能夠起到支持造血的作用,而GVHD的發(fā)生與供者造血干細(xì)胞在受者體內(nèi)的植入比例密切相關(guān),如何通體外處理降低供者造血干細(xì)胞的生長(zhǎng)、增殖活性,使其僅在受者體內(nèi)短時(shí)間、低比例植入,起到促進(jìn)受者自身造血恢復(fù),同時(shí)避免GVHD的發(fā)生是本課題重點(diǎn)要解決的問(wèn)題。 與其他途經(jīng)相比,體外照射是降低造血干細(xì)胞活性最為簡(jiǎn)單有效的方法,在第一部分的研究中我們,利用半相合小鼠供受者模型(CB6F1→BALB/c),將動(dòng)員后供者脾臟造血干細(xì)胞分別給與2.5Gy、5Gy、7.5Gy、10Gyγ射線照射后輸入6 Gy亞致死劑量輻射損傷的受者小鼠體內(nèi)(3x107/只),發(fā)現(xiàn)照射后供者造血干細(xì)胞能夠加快受者小鼠的造血恢復(fù),而且體外照射劑量越小,支持造血的作用越明顯。以2.5Gy照射細(xì)胞效果最好,5Gy、7.5Gy照射細(xì)胞次之,10Gy照射細(xì)胞對(duì)造血幾乎無(wú)明顯促進(jìn)作用。同時(shí)我們利用流式細(xì)胞儀聯(lián)合實(shí)時(shí)定量PCR的方法檢測(cè)供者細(xì)胞在受者小鼠體內(nèi)的植入情況發(fā)現(xiàn),2.5Gy照射供者造血干細(xì)胞在部分受者小鼠外周血中形成穩(wěn)定、高比例植入(95%),從而取代受者造血,引發(fā)GVHD導(dǎo)致小鼠死亡。而5Gy照射造血干細(xì)胞且僅在受者外周血中形成微量植入/嵌合(10-3-10-4水平),持續(xù)2-4月后被受者完全被排掉,觀察至術(shù)后半年,無(wú)一例小鼠發(fā)生GVHD。照后1月,未在受者外周血中檢測(cè)到經(jīng)7.5Gy、10Gy照射的供者細(xì)胞成分,兩組小鼠同樣無(wú)GVHD發(fā)生。通過(guò)上述實(shí)驗(yàn)我們發(fā)現(xiàn)7.5Gy、5Gy照射供者造血干細(xì)胞輸注能夠起到既支持受者自身造血恢復(fù)又無(wú)GVHD發(fā)生的作用,為ARS的臨床治療提供了新思路。隨后我們將7.5Gy、5Gy照射供者造血干細(xì)胞與G-CSF聯(lián)合后觀察其對(duì)6 Gy亞致死劑量以及8Gy致死劑量輻射損傷小鼠的影響,發(fā)現(xiàn)聯(lián)合后對(duì)造血恢復(fù)有很好的協(xié)同作用,尤其是能夠顯著提高血紅蛋白及血小板的水平,而且能大幅提高8Gy輻射損傷小鼠生存,其中以5Gy照射細(xì)胞聯(lián)合G-CSF效果最為明顯,顯著優(yōu)于單獨(dú)使用G-CSF(70+10.1% VS 30+10.1%)。植入檢測(cè)發(fā)現(xiàn),7.5Gy照射細(xì)胞在8Gy輻射損傷小鼠體內(nèi)形成微量嵌合,2-4個(gè)月內(nèi)被受者排掉。5Gy照射細(xì)胞在8Gy輻射損傷小鼠體內(nèi)的植入比例差別較大,從微量嵌合到高比例植入,但均不穩(wěn)定,2-4個(gè)月內(nèi)被排掉,均恢復(fù)受者小鼠自身造血,兩細(xì)胞治療組無(wú)一例發(fā)生GVHD。這對(duì)于重度骨髓型放射病有極少量殘存自身造血干細(xì)胞患者的臨床治療有重要參考價(jià)值。我們繼續(xù)將上述劑量體外照射的造血干細(xì)胞與G-CSF聯(lián)合用于超大致死量10Gy輻射損傷小鼠的救治。結(jié)果發(fā)現(xiàn),7.5Gy照射造血干細(xì)胞+G-CSF組、G-CSF組以及對(duì)照組小鼠全部死亡,而5Gy照射造血干細(xì)胞+G-CSF組小鼠存活率達(dá)到60%,造血快速恢復(fù),但5Gy照射供者造血干細(xì)胞在受者小鼠體內(nèi)形成高比例穩(wěn)定嵌合,快速的造血恢復(fù)來(lái)源于供者細(xì)胞的完全植入取代受者造血,而并非受者自身造血恢復(fù),因此導(dǎo)致部分小鼠發(fā)生慢性GVHD死亡,提示在受者小鼠無(wú)殘存自身造血的情況下,體外照射的造血干細(xì)胞治療可能存在的風(fēng)險(xiǎn)。在第一部分中我們還就照射后造血干細(xì)胞體外增殖能力、體內(nèi)歸巢以及其支持造血的機(jī)理進(jìn)行了初步研究,并在正文中對(duì)造血干細(xì)胞體外滅活程度、受者免疫抑制程度、供者細(xì)胞植入比例與GVHD發(fā)生關(guān)系進(jìn)行了詳細(xì)分析。 在第二部分的研究中,我們選取在第一部分中能夠起到良好造血促進(jìn)作用的5Gy照射供者造血干細(xì)胞與G-CSF聯(lián)合方案,觀察其對(duì)6Gy亞致死量輻射損傷小鼠免疫功能的影響。結(jié)果提示聯(lián)合治療能夠調(diào)整輻射損傷小鼠中樞及外周淋巴細(xì)胞亞群的比例,促進(jìn)淋巴細(xì)胞功能恢復(fù)。G-CSF聯(lián)合細(xì)胞輸注后可促進(jìn)胸腺雙陰性細(xì)胞的增殖、分化,減輕雙陽(yáng)性細(xì)胞凋亡,促進(jìn)其向CD4、CD8單陽(yáng)性細(xì)胞分化,提高外周血淋巴細(xì)胞絕對(duì)值,以及CD3+CD4+、CD3+CD8+細(xì)胞比例,增強(qiáng)T、B淋巴細(xì)胞對(duì)絲裂原的反應(yīng)。利用實(shí)時(shí)定量PCR對(duì)胸腺TRECs水平檢測(cè)我們發(fā)現(xiàn)照后30d、60d,G-CSF聯(lián)合細(xì)胞輸注組TRECs拷貝數(shù)明顯高于對(duì)照組及單獨(dú)使用G-CSF組,流式細(xì)胞儀檢測(cè)受者小鼠外周初始T細(xì)胞水平發(fā)現(xiàn)聯(lián)合治療組外周血中初始T細(xì)胞水平明顯增高,提示聯(lián)合治療可增加胸腺近期輸出功能,促進(jìn)輻射損傷后中樞免疫功能重建。 通過(guò)上述實(shí)驗(yàn)研究,我們發(fā)現(xiàn)G-CSF聯(lián)合體外一定劑量照射后半相合異基因造血干細(xì)胞輸注能夠促進(jìn)亞致死量及有極少殘存造血的致死量輻射損傷小鼠自身造血及免疫功能恢復(fù),無(wú)GVHD等副反應(yīng)發(fā)生,為ARS的細(xì)胞治療提供了一條新思路、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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

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本文編號(hào)：2047059