人脐带间充质干细胞来源胞外囊泡在异基因造血干细胞移植中的作用及机制

发布时间：2018-06-21 07:41

本文选题：间充质基质细胞 + 胞外囊泡　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：研究背景目前,异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation, allo-HSCT)是某些恶性血液病的唯一治愈方法。急性移植物抗宿主病(acute graft-versus-host disease,aGVHD)是allo-HSCT后受者死亡的主要原因。迄今为止尚无最佳aGVHD预防方法,亟需开发更有效的预防方案。间充质干细胞(mesenchymal stem cells, MSCs)因其独特的免疫调节及组织修复能力,成为allo-HSCT过程中细胞疗法的理想“候选者”。大量临床试验提示过继性输注MSCs能够预防allo-HSCT后致死性aGVHD的发生,并且能够促进造血重建。然而,MSCs的多向分化能力及其体内长期生存活性对受者远期影响不明,出于安全性考虑限制了MSCs的广泛应用。近年来研究提示胞外囊泡(extracellular vesicles, EVs)介导的旁分泌机制在MSCs为基础的治疗中发挥关键作用,并且越来越多的临床前及临床试验提示在组织修复及免疫调节领域,MSCs来源的外泌体及微泡有望替代MSCs发挥作用。研究目的本研究拟探讨人脐带间充质干细胞来源胞外囊泡(EVs released from human umbilical cord derived MSCs,hUC-MSC-EVs)在allo-HSCT中是否可以预防aGVHD和促进造血重建及可能的机制。研究方法及结果①Allo-HSCT小鼠模型的建立。BALB/c小鼠7.5Gy X射线全身照射后4-6小时内输注C57BL/6小鼠6×106骨髓细胞及3×106脾细胞。移植后21天左右受鼠出现明显的体重下降、活动度减少、弓背、竖毛、皮肤裸露等aGVHD临床表现。相应地,移植后1个月aGVHD靶器官：皮肤、肝脏、肠道出现典型的组织形态学改变。移植后约两个月,受鼠全部死于严重的aGVHD。② hUC-MSC-EVs的分离及鉴定。hUC-MSCs在无血清培养基中培养48小时后收集细胞培养上清,2000g离心30分钟以去除细胞及碎片。上清给予两次100000g,2小时,4℃超速离心。PBS重悬最终EVs沉淀。BCA法测定hUC-MSC-EVs蛋白浓度。透射电子显微镜观察hUC-MSC-EVs是一群直径介于30到100纳米大小的异质性球状体。③探究hUC-MSC-EVs对allo-HSCT小鼠aGVHD的预防作用。allo-HSCT第0天、第7天分别输注200μg hUC-MSC-EVsohUC-MSC-EVs输注组受鼠aGVHD临床表现减轻,靶器官组织形态学改变减轻,死亡率降低。淋巴细胞亚群检测提示hUC-MSC-EVs输注组受鼠CD3+CD8+T细胞比例及绝对值明显降低,但CD3+CD4+/CD3+CD8+T细胞比例升高。此外,hUC-MSC-EVs输注组受鼠促炎细胞因子：IL-2、TNF-α及IFN-y水平降低,抗炎细胞因子：IL-10水平增高。与此一致,体外实验提示hUC-MSC-EVs处理组丝裂原诱导的小鼠淋巴细胞增殖受抑,且细胞因子变化模式与体内实验相似。④探究hUC-MSC-EVs对allo-HSCT后小鼠造血重建的影响。allo-HSCT第0天输注200μg hUC-MSC-EVs。hUC-MSC-EVs输注组小鼠外周血象恢复时间缩短。体外实验发现hUC-MSC-EVs处理组小鼠骨髓细胞各系集落形成率增加。研究结论①成功建立了allo-HSCT小鼠模型;②差异离心法是获取hUC-MSC-EVs的有效方法;③ hUC-MSC-EVs通过调节免疫反应预防allo-HSCT后aGVHD的发生;④ hUC-MSC-EVs促进allo-HSCT后造血重建。
[Abstract]:Background, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only cure for some malignant hematopathy. Acute graft-versus-host disease (acute graft-versus-host disease, aGVHD) is the main cause of death after allo-HSCT. So far, there is no best aGVHD preconditioning. Mesenchymal stem cells (MSCs) is an ideal candidate for cell therapy in allo-HSCT process because of its unique immunomodulatory and tissue repair ability. A large number of clinical trials suggest that adoptive infusion of MSCs can prevent the occurrence of fatal aGVHD in allo-HSCT after allo-HSCT. However, the multiple differentiation ability of MSCs and the long-term survival activity of the body and the long-term survival activity of the body are unknown, and the wide application of MSCs is limited out of safety considerations. In recent years, research suggests that the parapet mechanism mediated by extracellular vesicles (EVs) plays a key role in MSCs based treatment. Use, and more and more preclinical and clinical trials are suggested in the field of tissue repair and immunomodulation. The exocrine and microbubbles of MSCs sources are expected to replace MSCs. The purpose of this study is to explore the origin of extracellular vesicles derived from human umbilical cord mesenchymal stem cells (EVs released from human umbilical cord derived MSCs, hUC-MSC-EVs) in allo Whether or not -HSCT can prevent aGVHD and promote hematopoiesis and possible mechanism. Research methods and results (1) the establishment of Allo-HSCT mice model of.BALB/c mice after 7.5Gy X ray whole body irradiation of C57BL/6 mice within 4-6 hours of infusion of 6 x 106 bone marrow cells and 3 x 106 spleen cells. After 21 days of transplantation, the mice were exposed to significant weight loss and activity degree. AGVHD clinical manifestations, such as reduction, arbor, erect hair, skin exposure, etc. correspondingly, 1 months after transplantation, aGVHD target organs: skin, liver, and intestines have typical histomorphological changes. About two months after transplantation, all rats died of severe aGVHD. (2) hUC-MSC-EVs separation and identification, and.HUC-MSCs was collected in serum-free medium after 48 hours of culture. Cell culture supernatant, 2000g centrifugation for 30 minutes to remove cells and debris. The supernatant was given two times 100000g, 2 hours, 4 C overspeed centrifugation.PBS suspension final EVs precipitation.BCA method for the determination of hUC-MSC-EVs protein concentration. The transmission electron microscope observation hUC-MSC-EVs is a group of heterogeneous spheroids with a diameter of 30 to 100 nanometers. (3) explore hUC-MSC-EVs pairs The preventive effect of aGVHD in allo-HSCT mice was.Allo-HSCT for zeroth days, and on the seventh day, the clinical manifestations of aGVHD in the 200 mu g hUC-MSC-EVsohUC-MSC-EVs infusion group were relieved, the morphological changes of the target organs were reduced and the mortality was reduced. The lymphocyte subgroup detection suggested that the proportion and absolute value of CD3+CD8+T cells in the hUC-MSC-EVs infusion group decreased significantly. The proportion of CD3+CD4+/CD3+CD8+T cells increased. In addition, the level of IL-2, TNF- alpha and IFN-y decreased in the hUC-MSC-EVs infusion group, and the level of anti-inflammatory cytokines: IL-10 increased. In vitro experiments, it was suggested that the proliferation of lymphatic cell proliferation induced by mitogen in hUC-MSC-EVs treated mice was inhibited, and the pattern of cytokines changes in the body and the body were in the body. The effect of hUC-MSC-EVs on the hematopoietic reconstitution of mice after allo-HSCT was studied. The recovery time of peripheral hemogram of mice in the zeroth day infusion group of 200 mu g hUC-MSC-EVs.hUC-MSC-EVs infusion group was shortened. The colony formation rate of each mouse bone marrow cell in the hUC-MSC-EVs treatment group was added in vitro. The conclusion was that (1) a successful establishment of allo-HSCT was established. The mouse model; differential centrifugation method is a effective method to obtain the hUC-MSC-EVs; the hUC-MSC-EVs aGVHD by regulating the immune response after allo-HSCT and hUC-MSC-EVs promote prevention; reconstruction after hematopoietic allo-HSCT.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R457.7

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