【作者】 田登梅；

【导师】 梁英民； 韩骅；

【作者基本信息】 第四军医大学 ， 内科学， 2013， 博士

【摘要】 目前造血干细胞移植已经成为临床治疗血液病和血液系统恶性肿瘤的重要手段之一，其中骨髓、外周血和脐血是其造血干细胞的重要来源。由于脐血来源丰富、采集方便、免疫原性弱，又较骨髓和外周血移植有更多优势，因此，近年来脐血移植越来越受到重视。然而，由于脐血造血干/祖细胞含量较低，难以满足成年及体重较大患者的需要；且脐血移植后造血重建延迟、感染性并发症增加，很难收到满意的疗效，已严重限制其在临床中的应用。如何有效地获取足量的造血干细胞以满足临床需要，已成为一个亟待解决的问题。大量的研究表明，Notch信号途径是调控干细胞增殖分化的重要信号通路之一，Notch受体和配体在造血系统广泛表达，激活Notch信号通路能够促进HSCs的自我更新，这为有效获取足量的HSCs提供了新的思路。本课题以人脐静脉内皮细胞作为培养体系的支持细胞，模拟造血干细胞生长的微环境，联合应用外源性生长因子SCF、TPO、FL、IL-6、IL-3及一种内皮细胞靶向的Notch配体D1R(人D1R，hD1R），与造血干/祖细胞共培养，建立一种有效的体外扩增体系，并通过给照射小鼠体内直接注射D1R蛋白，重点观察了其对HSPCs在骨髓、肝脏的植入和对造血微环境的影响，进一步分析了内皮细胞靶向的Notch配体激活的Notch信号在维持造血干/祖细胞自我更新、促进植入的重要作用及相关机制。研究结论如下：1、建立了基于Notch信号调节的体外培养体系，明显地扩增了小鼠的造血干/祖细胞，并通过体内移植实验证明了扩增的造血细胞具有长期造血重建的能力。提示D1R激活的Notch信号维持了HSPCs自我更新的特性，促进了增殖。2、在照射小鼠造血损伤实验中，同时给照射小鼠体内注射D1R融合蛋白，有效地保护了造血微环境，显著地扩增了体内残存的HSPCs，并通过骨髓移植实验证明扩增的体内HSPCs具有造血重建的能力。3、在小鼠骨髓移植实验中，直接给移植后的受体小鼠注射D1R融合蛋白，有效地促进了供体细胞在骨髓、肝脏、脾脏的植入，并通过在肝脏、脾脏诱导形成血管龛样的结构支持扩增了供体细胞，提高小鼠存活率，并且降低了小鼠造血重建所需的最低造血细胞数量。4、利用已建立的体外培养体系，有效地扩增了人脐血CD34+细胞，并通过NOD/SCID小鼠体内移植实验证明了扩增的造血细胞具有造血重建的能力。综上所述，我们通过内皮细胞靶向的Notch配体（D1R、 hD1R）建立了有效的体外扩增体系，并首次将D1R融合蛋白直接在小鼠体内应用，重点观察了其对HSPCs的植入和对造血微环境的影响，进一步明确了血管内皮细胞靶向的Notch配体激活的Notch信号对维持造血干/祖细胞特性、促进植入的重要作用，为实现临床应用提供实验基础。更多还原

【Abstract】 Recently, hematopoietic stem cell transplantation（HSCT）has become an importanttherapy for patients with hematonosis and hematologic malignancy.Umbilical cord blood(UCB), marrow and mobilized peripheral blood have been used successfully as principlesources of HSCT.UCB has become more attractive for its abundance, ready availabilityand immature immunogenicity compared to marrow or mobilized peripheral blood.Butumbilical cord blood transplantation (UCBT) in adults and high weigh patients is prevented by a limited number of hematopoietic stem cells (HSC) in each graft, whichleads to delayed engraftment and increased rates of infectious complications.Strategies toget sufficient HSCs for transplantation have been an important problem demandingprompt solution.A growing body of work has identified that the Notch pathway plays a fundamentalrole in regulating stem cell fate decisions to self-renew or differentiate.Notch receptorsand ligands are widely expressed in hemopoietic system.Notch activation is able tomaintain self-renewal of HSCs,which will be a novel thought to gain sufficient HSCs.Wehave developed an ex vivo expansion system for hematopoietic stem and progenitor cells(HSPCs) that used human umbilical vein endothelial cells (HUVECs) as feeder cells tomimic hematopoietic microenvironment supplemented with cytokines(SCF,FL,TPO,IL-6,IL-3) and endothelial cells-targeted Notch ligand （D1R,humanD1R）.Importantly, through in vivo D1R administration we assessed its influence onHSPCs engraftment in the bone marrow, the liver and hematopoietic microenvironment,and further analyzled the related mechanisms of Notch activation by theendothelium-targeted D1R in keeping self-renewal of HSCs and promoting engraftment.The main finds are as follows:1. The Notch-mediated ex vivo expansion system has been established and resulted in amarked increase in mouse HSPCs capable of enhanced long-term repopulation in themarrow.These suggested that Notch activation by D1R maintain the “stemness” of HSCsand promote expansion.2. Systemic administration of D1R to irradiated mice protected marrowmicroenvironment from the irradiation and caused a pronounced expansion of remnantbone marrow stem and progenitor cells in vivo, which were able to repopulate the marrow.3. D1R administration effectively promoted HSCs engraftment in both bone marrowand liver of mice receiving BM transplantation.Futher study found the formation ofvascular niche-like structures in the liver of recipient mice, which resulted in donor HSPCsexpansion, improved mouse survival and decreased the lowest effective transplant cellsdose keeping hematopoietic reconstitution. 4. Treatment of human cord blood CD34+cells with hD1R also substantially increasedcell counts in the culture system,which were capable of enhanced repopulation in themarrow of immunodeficient nonobese diabetic–severe combined immunodeficient(NOD-SCID) mice.In conclusion, the Notch-mediated ex vivo expansion system has successfully beenestablished and markedly increased HSPCs.We first used endothelial cells-targeted Notchligand D1R in vivo and observed its influence on HSPCs engraftment and hematopoieticmicroenvironment. Furthermore, we confirmed the role of Notch activation by using anendothelium-targeted Notch ligand in maintaining self-renewal of HSCs and promotingengraftment.更多还原