脐血CD34^+造血干细胞来源的树突状细胞体外培养体系的优化  

Improvement of the culture system in vitro for dendritic cells derived from cord blood CD34^+ hematopoietic precursor cells (HPC)

作　　者：顾镭[1] 邢美芬[1] 季晓辉[1] 孙志达[1] 杨晓帆[1] 王慧娟[1] 张明顺[1] 

机构地区：[1]南京医科大学微生物与免疫学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2006年第10期913-917,F0002,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省自然科学基金(BK2004148)资助项目

摘　　要：目的:优化脐血CD34+造血干细胞(HPC)来源的树突状细胞(DCs)诱导培养方法,为体外研究CD34+HPC诱导产生功能性树突状细胞(DCs)的影响因素奠定基础。方法:淋巴细胞分离液(Ficoll)分离获得脐血单个核细胞,免疫磁珠阳性分选CD34+HPC,并用流式细胞术鉴定CD34+HPC纯度;比较GT(GM-CSF+TNF-α)方案和GTI(GM-CSF+TNF-α+IL-4)方案及GTI方案中TNF-α和IL-4不同时段加入对诱导培养产生的DCs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DCs激发异体T细胞增殖能力。结果:免疫磁珠阳性分选CD34+HPC纯度可达90%以上;将CD34+HPC按GT方案和GTI方案进行培养,均可诱导产生DCs,但经GT方案诱导的DCsCD14表达较高,CD80、CD86、CD83、CD1a表达不如经GTI方案诱导的高;而GTI方案中,以TNF-α0h加入、IL-448h加入诱导培养的DCs各表型表达相对较佳,并具有激发异体T细胞增殖能力。结论:CD34+HPC经过合适的培养体系能够诱导分化为功能性DCs,以GM-CSF与TNF-α0h加入、IL-448h加入的GM-CSF+TNF-α+IL-4方案更为可取。Objective： To investigate the influential factors and provide improved method for inducing functional dendritic cells （DCs） derived from cord blood CD34^＋ hematopoietic precursor cells （HPC） in vitro. Methods ： CD34＋HPC were isolated and purified from umbilical cord blood by using a high-gradient magnetic cell sorting system （MACS）. CD34＋ cells were cultured with different inducing medium which contained different cytokines： GM-CSF and TNF-α （GT） or GM-CSF, TNF-α and IL-4 （GTI） ,in order to differentiate into DCs. DCs derived from CD34＋HPC were identified according to their morphology, phenotypes by FACS and laser scanning eonfoeal fluorescence microscopy. Their abilities of inducing proliferation of allogenic T cells were assessed 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-α and IL-4. DCs induced with GTI expressed higher level of surface antigen of CD80, CD86, CD83, CDIa and lower level of CDI4 than those induced with GT. DCs showed typical morphol- ogy and property when they were induced with proper cytokines of GM-CSF, TNF-α added at 0 h and IL-4 added at 48 h. Conclusion ： DCs could be generated from CD34^＋HPC by proper culture system. The culture system of GM-CSF ＋ TNF-α ＋ IL-4 （GM-CSF and TNF-α were added at 0 h, and IL-4 was added at 48 h） is preferred.

关 键 词：脐血 CD34^+HPC 树突状细胞 体外培养 

分 类 号：R392.12[医药卫生—免疫学]