中文摘要：

脐带血间充质干细胞（umbilical cord blood mesenchymal stem cells，UCB-MSCs）不仅可以作为滋养层细胞支持造血干细胞在体外的大规模扩增，在造血移植过程中还能够降低并发症的发生率以及加速造血重建功能的恢复．但是，目前UCB-MSCs的原代分离培养成功率一般只有30％左右，为进一步提高该成功率，利用正交实验方法对UCB-MSCs体外培养的主要影响因素：细胞的接种密度、细胞因子的组合及用量、是否添加血清和滋养层细胞，进行逐层筛选，并对培养出的间充质干细胞进行了流式细胞分析和向成骨、软骨及脂肪方向的诱导分化检测，以期获得UCB-MSCs培养的最佳方法．实验结果表明，细胞的接种密度是UCB-MSCs培养最显著的影响因素（P〈0．1），接种密度越大，MSCs越容易生长，能够培养出MSCs的几率就越大，其次为细胞因子，添加细胞因子能有效地刺激MSCs的生长．在高接种密度的基础上，添加细胞因子IL-3（15μg／L）和GM-CSF（5μg/L），可大大提高UCB-MSCs体外原代培养的成功率，从30％左右提高到90％以上．流式细胞检测结果显示，所分离培养的细胞表达间充质干细胞的抗原（CD13^＋、CD29^＋、CD44^＋、CD105^＋、CD166^＋），不表达造血细胞的抗原（CD34^-、CD45^-、HLA-DR^-），并能够向成骨、软骨及脂肪方向分化，这与源于骨髓的间充质干细胞相一致．所建立的培养方法能够为UCB-MSCs的临床应用提供大量优质的种子细胞．

英文摘要：

Mesenchymal stem cells （MSCs） derived from umbilical cord blood （UCB） can not only support hematopoietic stem cells （HSCs） expansion in vitro as stromal cells, but also alleviate complications and accelerate the recovery of hematopoiesis during hematopoietic stem cell transplantation. The ratio of successful isolation and culture of UCB-MSCs, however is only about 20%-30% to date. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In order to improve this ratio and optimize the culture method for UCB-MSCs, factorial design were applied to investigate the main influencing factors, including cell inoculate density （ID）, combination and dose of cytokines, presence of serum and stromal cells or not. The experimental results indicated that ID was the most significant influencing factor for UCB-MSCs culture when P 〈 0.1. Higher ID leaded to higher probability of success and better growth for UCB-MSCs. Then were cytokines, which could stimulate the growth of UCB-MSCs effectively. Based on the high cell ID, the optimized culture condition was determined with cytokines IL-3 （15 μg/L） and GM-CSF （5 μg/L） added in the traditional medium of MSCs. Then the probability of obtaining UCB-MSCs can be increased up to 90% from 30% with traditional culture method. Moreover, the characteristics of UCB-MSCs were tested by flow cytometric analysis and multi-lineage differentiation identification for osteoblast, chondrocyte and adipocyte. The results showed that the fibroblast-like cells expressed MSCs surface markers of CD13, CD29, CD105, CD166 and CD44 positively and CD34, CD45 and HLA-DR negatively. Meanwhile the cells could differentiate into osteoblasts, chondrocytes and adipocytes similarly to MSCs derived from bone marrow. In conclusion, an efficient protocol have been developed to culture UCB-MSCs by adding cytokines IL-3 （15 μg/L） and GM-CSF （5 μg/L）.