摘要
本研究探讨脐血CD133+(UCB-CD133+)细胞体外扩增巨核系祖细胞的能力和最佳收获时间。采用免疫磁珠激活细胞分选系统(MACS)分选UCB-CD133+细胞,将纯化的UCB-CD133+细胞接种于含TPO、IL-3和SCF的无血清液体培养体系中体外扩增巨核系祖细胞,在培养第7、10和14天进行细胞计数,流式细胞仪检测扩增过程中CD133、CD34、CD41抗原表达的动态变化,并采用半固体法对不同扩增阶段的细胞进行巨核祖细胞集落形成单位(CFU-MK)培养。结果表明:培养至第7天时,UCB-CD133+细胞扩增效果最佳,扩增了8.2±2.2倍;培养至第14天,细胞总数扩增了116倍;培养至10天时,平均1个CD133+细胞所产生的CD133+CD41+和CD34+CD41+细胞数最多,分别为2.5±0.9和2.6±0.5个,所产生的CD41+细胞为20.3±5.9个;扩增前后的UCB-CD133+细胞均能形成CFU-MK,扩增第10天的UCB-CD133+细胞所形成的CFU-MK总数最多,CFU-MK扩增倍数为59.5±11.8倍。巨核细胞免疫组织化学染色显示,扩增后的巨核系细胞多呈幼稚状态,未见血小板形成。结论:UCB-CD133+细胞具有较强的体外扩增巨核系祖细胞的能力,培养第10天扩增效能最佳。
To study the expansion potentiality of megakaryocyte progenitor cells （MPCs） derived from human umbilical cord blood CD133^＋ （ UCB-CD133^＋ ） cells and determine the optimal harvest time. UCB-CD133^＋ cells were purified from mononuclear cells （MNCs） by magnetic activated cell sorting （MACS） and seeded in serum-free liquid culture medium supplemented with thrombopoietin （ TPO）, interleukin-3 （ IL-3 ） , and stem cell factor （SCF） to expand MPCs. At day 0, 6, 10 and 14 of culture , the total cell number was counted and the dynamic changes of CD133, CD34, and CD41 antigen expression during ex vivo expansion were analyzed by flow cytometry （FCM）. At different expansion times, the CD133^＋ cells were collected and cultured in collagen semisolid medium to carry out CFU-MK colony culture. The incidence of CFU-MK was calculated and the morphology of MPCs and CFU-MK were detected by immunohistochemisa-y and Wright-Giemsa staining. The results showed that UCB-CD133^＋ cells optimally expanded at day 7 with expansion multiple of 8.2 ± 2.2 in serum-free liquid culture systems and the total cell number was expanded by 116-fold at day 14. At 10 days, each UCB-CD133 ^＋ cell can form 2.5 ± 1.0, 2.6 ± 0.5 and 20.3 ± 5.9 cells of CD133 ^＋CD41 ^＋ ,CD34 ^＋ CD41 ^＋ and CD41 ^＋ repectively, from which the number of CD133^＋ CD41 ^＋ and CD34 ^＋ CD41 ^＋ cells reach the highest. UCB-CD133 ^＋ cells both before and after expansion could form CFU-MK, the total number of CFU-MK reached the peak from cells of 10 days expansion of UCB-CD133 ^＋cells and the expansion multiple of CFU- MK was 59.5^＋11.8. Immunohistochemical results indicated that the expanded megakaryocytic cells were immature and no sign of platelet formation. It is concluded that the human UCB-CD133^＋ cells have a high abilitity of MPC expansion, 10 days of culture can be result in optimal expansion effect.
出处
《中国实验血液学杂志》
CAS
CSCD
2008年第3期645-649,共5页
Journal of Experimental Hematology
基金
贵州省科技计划发展项目,编号2051
2003JGY005
关键词
CD133^+细胞
造血干/祖细胞
巨核系祖细胞
体外扩增
脐血
CD133^＋ cell
hematopoietic stem/progenitor cell
megakaryocyte progenitor cell
ex vivo expansion
umbilical cord blood
