肿瘤坏死因子α和白细胞介素4对脐血CD34+造血干细胞来源的树突状细胞体外培养体系的影响

背景：造血干细胞是构筑免疫系统的最早的细胞，能分化为多种细胞，其中具有包括免疫应答调控树突状细胞。树突状细胞的诱导培养因前体细胞来源不同，所采用的细胞因子，及最佳的细胞因子配伍、应用顺序、实验室培养条件亦不相同，树突状细胞的发育、各种表型的表达及成熟度也不尽相同。目的：观察肿瘤坏死因子α和白细胞介素4对脐血CD34＋造血干细胞来源的树突状细胞诱导培养体系的影响，探寻该培养体系优化方法。设计、时间及地点：观察性实验，于2005—03／11在南京医科大学微生物与免疫学实验室完成。材料：健康新生儿脐血为南京市八一医院产妇同意捐赠。CD34单克隆抗体-磁珠分离系统为德国MiltenyiBiotec公司产品；重组人粒细胞巨噬细胞集落刺激因子（GM—CSF）、重组人白细胞介素4和重组人肿瘤坏死因子α为美国PeproTech公司产品。方法：淋巴细胞分离液分离获得脐血单个核细胞，免疫磁珠阳性分选CD34＋造血干细胞，并用流式细胞术鉴定CD34＋造血干细胞纯度；比较GT（GM-CSF＋肿瘤坏死因子α）方案和GTI（GM-CSF＋肿瘤坏死因子α＋白细胞介素4）方案及GTI方案中肿瘤坏死因子α和白细胞介素4不同时段加入对诱导培养产生的树突状细胞成熟的影响；通过激光共聚焦显微镜观察细胞形态，流式细胞仪分析细胞表型及3H-TdR检测树突状细胞激发异体T细胞增殖能力。结果：免疫磁珠阳性分选CD34＋造血干细胞纯度可达90％以上。将CD34＋造血干细胞按GT方案和GTI方案进行培养，均可诱导产生树突状细胞，CD34的阳性表达率逐渐下降，HLA-DR的表达下降（P〈0．05），树突状细胞的相关分化抗原CD80，CD86，CD83和CDla的表达均相应增加，培养13～15d的细胞各表型表达较7-9d，10～12d充分。但经GT方案诱导的树突状细胞CD14表达较高，CD80，CD86，CD83，CD1α表达不如经GTI方案诱导的高；而GTI方案中，以肿瘤坏死因子Q0h、白细胞介素448h加入诱导培养的树突状细胞各表型表达相对较佳，其细胞表达CD80，CD86均较其他组高，尤以CD86表达为著，并具有激发异体T细胞增殖能力。结论：CD34＋造血干细胞经过合适的培养体系能够诱导分化为功能性树突状细胞，以GM-CSF与肿瘤坏死因子α0h加入、白细胞介素448h加入的GM-CSF＋肿瘤坏死因子α＋白细胞介素4方案更为可取。

Influence of tumor necrosis factor-alpha and interleukin-4 on the in vitro culture system for dendritic cells derived from cord blood CD34+hematopoietic precu rsor cells

BACKGROUND: Blood stem cells (BSCs) are the most primitive cells in the immune system and can be differentiated into many kinds of cells. As the regulatory cells of immune response, dendritic cells (DCs) have been attracted more and more attention in the field of autoimmune diseases. Due to different resources of precursor cells, DCs have different cytokines, ideal cytokine matching,applying orders, and experimental cultured conditions. Furthermore, development, phenotypic expression, and mature degree are still different.OBJECTIVE: To investigate the influence of tumor necrosis factor- α (TNF- a ) and interleukin-4 (IL-4) on the culture system and provide improved method for inducing functional DCs in vitro, derived from cord blood CD34+ hematopoietic precursor cells (HPC).DESIGN, TIME AND SETTING: Observational study, which was performed in the Department of Microbiology and Immunology, Nanjing Medical University from March 2005 to November 2005.MATERIALS: The cord blood was collected from neonatal umbilical cord in the 81 Hospital of Nanjing City. CD34 monoclonal antibody coated magnetic bead system (MACS) was provided by Miltenyi Biotec Company, Germany; human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF), human recombinant interleukin-4 (rhlL-4), and human recombinant tumor necrosis factor-α (rhTNF-α) by Pepro Tech Company, USA.METHODS: CD34+HPC were isolated and purified from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Then the cells were cultured with different culture medium which contained different combinations of cytokines:GM-CSF and TNF-α (GT) or GM-CSF, TNF-α and IL-α (GTI) in order to be induced to differentiate into dendritic cells (DCs).The DCs derived from CD34+HPC were identified for their morphology and phcnotype by FACS and laser scanning con_focal fluorescence microscopy, and also for their abilities of inducing proliferation of allogenic T cells by 3H-TdR incorporation assay.RESULTS: The purity of selected CD34+ cells with MACS was more than 90%. DCs could be obtained from CD34+HPC by the culture in presence of GM-CSF and TNF-α or GM-CSF, TNF- o and IL-4. With the time of culture lasting, the cells expressed lower level surface antigen of CD34 and HLA-DR (P ＜ 0.05), and possessed the phenotypes of DCs characterized by higher expression of CD80, CD86, CD83 and CDIa. At 13-15 days, the cells possessed higher level of phenotypes of DCs compared to 7-9 days and 10-12 days. DCs induced with GTI culture system expressed higher levels of surface antigen CD80, CD86, CD83,CD 1 a and a lower level of CD 14 than those induced with GT culture system. DCs displayed typical morphology and property and expressed higher level surface antigen of CD86 and CD80, especially expressed CD86 when they were induced with proper cytokines of GM-CSF and TNF- α added at 0 hour, and of IL-4 added at 48 hours.CONCLUSION: DCs can be generated from CD34+HPC by proper culture system, the design of GM-CSF + TNF- α + IL-4(GM-CSF and TNF- α were added at 0 hour, IL-4 was added at 48 hours) is preferred.