脐带血造血干细胞的分离、提纯与单个干细胞的培养

SEPARATION PURIFICATION IN HEMATOPOIETIC CELL UMBILICAL CORD BLOOD AND SINGIE STEM CELL CCULTURE

为了获得纯度较高的造血干细胞供临床应用,我们对人脐带血(Human Umbilical Cord Blood)简称脐血(CB)的分离、提纯和脐血单个干细胞的培养进行了研究,现摘要报导如下:取肝素抗凝的脐血用Ficoll分离获得低密度(LD)细胞,并将LD细胞采用贴壁方法获取非贴壁细胞(NAL)。周NAL细胞进一步作E—玫瑰花结形成试验,去除T淋巴细胞得到NALT^-细胞。然后用CD_(34)单抗作间接免疫荧光标记并用流式细胞仪分离提纯,最后得高标记的CD_(34)~#细胞。在含有甲基纤维素培养体系和重组的四种造血因子(rEPO、rhuMGF、rhull—3、rhuGM—CSF)的96孔板孔中,每孔加入1个CD_(34)~#细胞,在37°C5％CO_2的饱和湿度的二氧化碳培养箱培养14天观察结果。实验结果证明,序列分离提纯技术可大大浓集脐血造血干、祖细胞,集落产率达60～80％,平均为70％。与骨髓相比,脐血中含有丰富的造血干、祖细胞,主要含HPP-CFC,占集落总数的50％,其次是CFu-GM,CFu-GMM和BFU-E,因此脐血有较高的增殖活力。

In behalf of obtaining more pure hematopoietic stem cells for Clinical use. We had researched for separation, purification of human umbilical cord blood (CB) and its single stem cell culture we will sum marized as follw; After obtaining heparin anticoagulated CB,Low density cells were separated by cen-trifugation of using ficoll liquid to then and get nonadherent Low density (NAL)from LD cells by adher ent method. Then removed T-Lymphocyte from NAL by means of E-roset forming test, NALT, cells were harvested. After separation and purification with indirect immunofluorescent label on CD34 MoAd by flow cytometry, a high labelled CD34, cell were obtained. The cells were suspended in 96 well plate with Methycellulese culture system and four Kinds of recombinant hemopoietic factors. (rEPO rhuMGF rhuIL-3 rhuGM-CSF) ,One34 cell was added in each well. The culture was incubated at 37癈 in a humidi-tied atmosphere of 5% CO for 14 days,Them Watched the results this Experimental. Results proved that sequential separation and purification technique can largely concentrate hemopoietic stem and pro genitor cell in CB.Colony forming unit rate were achieved about 60-80 percent. Mean value 70% .Com pared with obtained from marrw material CB contains more rich hemopoietic stem and progenitor cells, In which, HPP-CFC accounts for 50 percent, The rest are CFU-GM,CFU-GEMM and BFU-E. There fore,CB has a of stronger reproducible activity.