摘  要

1.目的：高剂量放化疗会导致严重的中性粒细胞减少，在此期间患者具有很高的感染风险。虽然使用注射粒细胞集落刺激因子能够在一定程度上缓解和治疗中性粒细胞减少症，但是对于危重的病人特别是骨髓造血干细胞移植尚未恢复的病患来说使用粒细胞集落刺激因子却不能立即起到作用，必须使用中性粒细胞输注的方法对患者进行急救。但这种输注治疗所需要的大量的细胞来源成为了主要的限制因素，因捐献中性粒细胞的供体需要使用G-CSF来刺激外周血内中性粒细胞的数量，复杂的捐献过程严重的限制了这种方法可用性和嗜中性粒细胞来源。为了克服这些问题提供充足的中性粒细胞用于临床救治，在文献报道的基础上结合我们实验室在造血干细胞扩增方面的巨大优势，在降低生产成本及制备产业化可行性的推动下我们优化了G-CSF和GM-CSF两种细胞因子的表达和大规模制备过程，接下来我们实验了大规模离体高效扩增分化造血干细胞（HSC）为嗜中性粒细胞有效方案，并进一步确证了所获得的中性粒细胞具有与人体外周血分离的中性粒细胞的相同功效。

2.方法：使用免疫磁珠分选的方法从健康人脐带血（UCB）中分离CD34⁺造血干细胞，通过我们实验室的已经优化成熟的低成本基础培养基进行了多次的细胞组合因子优化实验，最终筛选出SFG3MT、SFG3M和SFG三种细胞因子组合来扩增和分化造血干细胞。同时我们在文献报道的基础上进一步实验优化将培养分成了四个阶段，每一阶段更换不同的细胞因子组合培养基，这样的培养方法是与细胞分化过程的阶段相吻合的。在筛选出优化的方案后我们在滚瓶培养系统上进行了平台条件优化，进一步调整了滚瓶培养系统分化的最佳条件和进行了规模化培养分化了临床移植级别的中性粒细胞。另外在研究过程中还使用原核细胞表达系统以包涵体的形式制备了造血干细胞扩增和中性粒细胞分化的重要细胞因子GM-CSF和G-CSF，建立了此蛋白实验室大规模制备的方法并进行了活性验证。为了保证获得中性粒细胞能够具有和人体细胞相同的功效我们在体外验证了它的杀菌功效。作为中性粒细胞最重要的功能趋化作用我们在体外和体内分别进行了验证，最后我们将离体分化获得的中性粒细胞和其祖细胞注射到了NOD/SCID中性粒细胞减少症小鼠体内，然后通过流式细胞术分析小鼠外周血和骨髓内的人体中性粒细胞的在小鼠体内分化和存活的状况并通过对小鼠生存状况的观察验证离体分化人中性粒细胞的安全性。

3.结果：通过使用我们实验室已经优化的造血干细胞扩增技术连同本实验中筛选的细胞因子组合及四阶段培养分化方法，使用本课题研究制备的GM-CSF和G-CSF两种重要细胞因子，在滚瓶培养平台上优化了高效的扩增和中性粒细胞定向分化条件，在培养18天后获得包含61.5％成熟嗜中性粒细胞的总细胞，脐带血造血干细胞数目总量获得了49000倍扩增。我们获得了的实验室制备相当于目前文献报道方法的5倍以上总细胞扩增效率，由一份脐带血造血干细胞获得的中性粒细胞足以能够提供12份中性粒细胞输注剂量。体外杀菌功能的实验发现在与体外分化中性粒细胞共同孵育后的细菌悬液涂布平板过夜培养后基本上无菌落形成，而未经与体外分化制备的中性粒细胞孵育的平板上则生长了大约200个菌落。体内和体外的趋化性试验表明离体的中性粒细胞能够对炎症因子具有趋化作用。由尾静脉注射人中性粒细胞经过16小时可以在炎症因子和酵母聚糖的作用下迁移到小鼠背部囊腔。有效性和安全性试验表明通过尾静脉注射NOD/SCID后4天后还能够在小鼠外周血内检测到人中性粒细胞的存在，这个存活时间是人体外周血分离的中性粒细胞一倍。另外使用体外培养分化9天的中性粒祖细胞尾静脉注射小鼠两个月后在小鼠的外周血内能够检测到人中性粒细胞的存在，还能够在骨髓内检测到人CD34⁺造血干细胞、中性粒祖细胞和成熟中性粒细胞。这一实验结论为中性粒细胞临床移植治疗提供了一个新的方案，因为在培养到第9天总细胞数目相比于初始造血干细胞已经扩增了900倍，细胞数量已经达到用于一个成年人用于移植治疗。输注的总细胞内因含有CD34⁺造血干细胞可以归巢到患者骨髓内持续产生成熟的血液细胞，并且第9天的培养细胞也可以冷冻保存和运输，有效的解决了成熟中性粒细胞不能冷冻保存运输的问题。更为重要的是输注中性粒祖细胞3个月后小鼠生存状态良好，未出现任何的发烧、过敏等不适症状也未有死亡小鼠，这也充分证明了使用我们的平台体外分化获得的中性粒细胞是安全和有效的，为临床输注有效治疗中性粒细胞减少症提供了新的材料和治疗方法。

4.结论：使用造血干细胞高效扩增技术和独特的低成本培养基配方我们已经建立了一个中试规模的体外扩增培养造血干细胞分化功能有效的人中性粒细胞系统。同时对于大规模制备过程中所需要的培养基及GM-CSF和G-CSF细胞因子都获得了独特的大规模制备技术，极大的降低了整个制备过程的生产成本，为产业化生产奠定基础。临床使用的一个嗜中性粒细胞输注单位（100ml）含有2×10¹⁰个细胞，来自一个人脐带血造血干细胞单位（80ml）的CD34⁺细胞使用我们的培养方法和平台将产生2.4×10¹¹个嗜中性粒细胞，这相当于12个单位/剂量的中性粒细胞可用于临床中性粒细胞输注。我们的培养成本也只有临床实验室进行同类实验成本的1/60，这种来源方便、安全有效、成本更容易为患者接受的方式对于临床大规模制备中性粒细胞用于输注治疗是非常有价值的。鉴于源自脐带血的大规模制备的嗜中性粒细胞的特征优异性，我们相信这些细胞可以用作临床中常规嗜中性粒细胞输血的有效替代来源。

Abstract

Large-Scale Ex vivo Generation of Human Neutrophils from Cord Blood CD34⁺ Cells

OBJECTIVE: High-dose radiotherapy and chemotherapy can lead to severe neutropenia, during which patients have a high risk of infection. Although the use of injection of granulocyte colony-stimulating factor can alleviate and treat neutropenia to a certain extent, the use of granulocyte colony-stimulating factor can not be used immediately for critically ill patients, especially can not be used in patients with bone marrow hematopoietic stem cell transplantation, we must use neutrophil infusion method of patients with first aid. However, the large number of cell sources required for this infusion treatment has become a major limiting factor because donor-derived neutrophils need to use G-CSF to stimulate the number of neutrophils in the peripheral blood, the complex donor process severely limiting the availability of this method and the source of neutrophils. In order to overcome these problems to provide adequate neutrophils for clinical treatment, based on the literature reported and on the basis of our laboratory in the stem cell expansion of the great advantages, We optimized the expression of G-CSF and GM-CSF and the large-scale preparation process, which was driven by the reduction of production cost and the feasibility of preparation. Next we describe an optimized four-stage culture approach using our in-house culture medium and the roller-bottle production platform that can generate neutrophils ex vivo on a large scale. We believe that our new stem cell expansion and differentiation platform is capable of providing large amounts of high quality neutrophils for clinical applications.

METHODS: CD34⁺ hematopoietic stem cells were isolated from healthy human umbilical cord blood (UCB) using magnetic bead sorting, and several cytokine combination optimization experiments were performed through our laboratory-optimized, low-cost basal medium. SFG3MT, SFG3M and SFG combination of three cytokines were used to expand and differentiate hematopoietic stem cells. At the same time, we further experimented on the basis of the literature to optimize the culture method into four stages, each stage to replace the different combination of cytokine cocktail culture medium, this culture method is consistent with the stage of cell differentiation process. In addition, the prokaryotic cell expression system was used to prepare the important cytokine GM-CSF and G-CSF of hematopoietic stem cell expansion and neutrophil differentiation in the form of inclusion bodies, and a large-scale preparation method of this protein laboratory was established. After optimizing the optimal cunlture system, and further adjusted the optimal conditions for the differentiation of the roller bottle culture system. In order to ensure that neutrophils can be obtained with the same function as human PB isolated nutrophils we have tested its bactericidal efficacy in vitro. As the most important functional chemotaxis of neutrophils we were verified in vitro and in vivo, and finally, we used in vitro differentiation of neutrophils and their progenitor cells injected into the neutropenia NOD/SCID mice, and then analysis of mouse peripheral blood and bone marrow neutrophils by flow cytometry, and to assess the safety of human neutrophils in vitro by observing the survival status of mice.

RESULTS: We optimized the efficient amplification and neutrophil targeting on the roller culture platform by using our laboratory-optimized hematopoietic stem cell growth technique combined with the cytokine cocktial and four-stage culture differentiation method selected in this differentiation study conditions. In addition, the use of subject self-prepared GM-CSF and G-CSF two kinds of cytokines, greatly reduce the large-scale preparation of neutrophils production costs.The total cells containing 61.5% mature neutrophils, the total number of umbilical cord blood hematopoietic stem cells was 49,000 folds of the amplification after 18 days of culture. The total cell expansion efficiency we obtained the experimental yield equivalent to more than 5 times the current literature reported, obtained neutrophils sufficient to be able to provide 12 neutrophil infusion doses by an umbilical cord blood hematopoietic stem cells. The in vitro bactericidal activity experiments have been found to be substantially colony-free after overnight incubation with bacteria-coated plates which co-incubated with in vitro differentiated neutrophils，no incubation with neutrophils resulted in 200 colonies grown on the plate. In vivo and in vitro chemotaxis studies have shown that neutrophils in vitro differentiated can chemotenize inflammatory factors. Human neutrophils were injected from the tail vein for 16 hours to migrate to the dorsal pouch of the mice under the action of inflammatory factors and zymosan. The efficacy and safety test showed that the presence of human neutrophils was detected in the peripheral blood of mice 4 days after the injection of NOD/SCID by tail vein, which was twice folds time of neutrophils isolated from human peripheral blood. In addition, the presence of human neutrophils was detected in the peripheral blood of mice two months after the administration of 9 days culture of neutrophil progenitor cells in tail vein, and human CD34⁺ hematopoietic stem cells, neutrophils progenitor cells and mature neutrophils were also detected in the bone marrow. This experimental conclusion provides a new regimen for clinical transplantation of neutrophils because the total number of cells grown to the 9th day has increased 900 folds compared to that of the initial hematopoietic stem cells and is sufficient for an adult to use for transplantation treatment. Infusion of the total cells containing CD34⁺ hematopoietic stem cells can homing to the patient's bone marrow to produce mature blood cells. The day 9 cultured cells can also be frozen and transported, effectively solve the mature neutrophils can not be frozen transport problems. More important that mice transplanted with neutrophil progenitor cells survived for three months, did not show any fever, allergies and other symptoms, and no death mice, which also fully demonstrated the use of our platform in vitro differentiation acquired neutrophils are safe and effective, providing new materials and treatments for the effective treatment of neutropenia for clinical infusion.

CONCLUSION: We have established a pilot scale in vitro to amplify hematopoietic stem cell differentiation functionally effective in human neutrophil system using hematopoietic stem cell high efficiency amplification techniques and unique low cost medium formulations. We have obtained a unique preparation technology for the large-scale preparation process required for the medium and GM-CSF and G-CSF cytokines at the same time, which laid the foundation for industrial production.This neutrophil source is convenient, safe and effective, the cost is easier for patients to accept, and for the clinical large-scale preparation of neutrophils for infusion therapy is very valuable. We believe that these cells can be used as an effective alternative source for routine neutrophil transfusion in view of the characteristic superiority of large-scale preparation of neutrophils derived from umbilical cord blood. Taken together, we have established a pilot-scale culture system to produce functional human neutrophils ex vivo. Considering that one neutrophil transfusion unit (100 ml) contains 2×10¹⁰ cells, the CD34⁺ cells from one UCB unit (80 ml) will generate 2.4×10¹¹ neutrophils, which are equivalent to 12 units/doses of neutrophils for clinical transfusion. Given excellent features associated with the neutrophils derived from UCB, we believe that these cells can be used as an alternative source to conventional neutrophil transfusion in the clinic.