人脐血CD_(34)^+细胞体外诱导树突状细胞及其功能的检测

Generation of dendritic cells derived from human cord blood CD_34^+ cells

目的 探讨人脐血CD+34 细胞体外诱导树突状细胞 (dendriticcells ,DCs)的可能性 ,并检测DCs的功能。方法 利用吸附单克隆抗体 磁珠分离系统 (MACS)获得脐血CD+34 细胞 ,体外以重组的人干细胞集落刺激因子 (hSCF)、人粒 巨噬细胞集落刺激因子 (hGM CSF)、人肿瘤坏死因子α(hTNF α)、人酪氨酸激酶受体家族III的配体 (hFL)诱生DCs。流式细胞仪检测DCs的表型 ,3H TdR掺入法检测DCs体外刺激同种异体T细胞增殖的功能 ,最后通过乳酸脱氢酶法检测DCs活化的T细胞对肿瘤细胞的杀伤性。结果 从人脐血分离到的CD+34 细胞 ,经hSCF、hGM CSF、hTNF α、hFL共同培养后 ,第 7天镜下即可见典型形态的DCs ,第 10天时明显增多 ,细胞表型检测见CD+1a 细胞的比例增加到 (2 8± 4) %(P <0 0 1) ,人类白细胞抗原DR(HLA DR)表达占 (94± 11) %。在三种不同的混和比例下 ,DCs均可刺激异源T细胞增殖 ,其中以DCs∶T细胞为 1∶5时增殖最明显。而且被激活的T细胞对三种不同组织来源的肿瘤细胞均产生了明显的杀伤性 (P <0 0 1)。结论 人脐血CD+34 细胞体外经细胞因子诱导培养 ,可生成大量具有典型功能的成熟DCs。

Objective Dendritic cells(DCs) are the most powerful professional antigen presenting cells in vivo as yet It has been indicated by foreign study groups that reimport of DCs pulsed with tumor associated antigen can induce an invigorating specific anti tumor response in clinic However, it is still difficult to obtain adequate DCs with simpleness and convenience in vitro The present study probed into the possibility of obtaining DCs from human cord blood CD + 34 cells and identified the functions of them Methods CD + 34 cells were isolated from human cord blood by mini MACS and cultured in a liquid culture system with hSCF、hGM CSF、hTNF α and hFL for 10 days Then the cells, many of which had the typical morphological properties of DCs observed by electron microscope, were analyzed for phenotypes of CD1a and HLA DR by FACS In order to identify their functions, DCs were tested for the capacity of stimulating the proliferation of allogeneic T cells by 3 H TdR incorporation and of initiating T cell dependent anti tumor immune responses by lactate dehydrogenase (LDH) kit Each of the above experiment was repeated for 10 times with 10 human cord blood samples Results In the presence of hSCF、hGM CSF、hTNF α and hFL , the cells with typical morphological properties of DCs at the 7th day were observed, much more obviously at the 10th day At that time, (28±4)% CD + 1a cells and (94±11)% HLA DR+cells were obtained In addition to these phenotypic properties, the induced DCs could remarkably stimulate the proliferation of allogeneic T cells with different ratios compared with control groups ( P <0 01), especially the CPM value increased from 362±43 to 2 814±285 when R:S was 5:1 Moreover, it was more significant that the T cells activated by DCs showed magnificent cytotoxicity on 3 different kinds of tumor cells compared with respective control groups ( P <0 01) In experimental groups, the highest killing rate was up to (39 6±2 9)% Conclusion Human cord blood could be served as a better source of mature DCs, which can function effectively