脐血间充质干细胞培养条件的优化

Optimal culture of human umbilical cord blood mesenchymal stem cells.

目的 探讨脐血间充质干细胞（mesenchymal stem cell，MSC）的培齐扩增条件。方法 取2004—10，2005—02广州华侨医院产科胎儿脐带血．比较不同的分离方法、接种密度、首次换液时间、不同的培养基对脐血中的间充质干细胞原代培养过程的影响，以流式细胞仪对优化条件下培养的间充质干细胞进行细胞周期分析，细胞表面标志检测，多种成分联合诱导其向脂肪、成骨方向分化，细胞化学染色检测诱导后的细胞变化。结果 在其他条件不变的情况下，羟乙基淀粉沉淀法优于Ficoll分离法，5×10^6／cm^2是脐血MSC培养的适宜接种密度，首次换液时间为7d.优化条件下培养的间充质干细胞不表达造血细胞系的标志（CDM／CD奶／CD14）及内皮细胞的标志CD106，强表达CD29、CD44、CD13，在适当的诱导每件下可向脂肪及成骨方向分化。结论 脐血中含有MSC．可以作为组织工程的种子细胞。

Objective To optimized the culture conditions of the human umbilical cord blood mesenchymal stem cells. Methods The umbilical cord blood was taken in Guangzhou Hua Qiao Hospital between Oct. 2004 to Feb. 2005. The isolation method, planting density, the time of the first medium changing, the influence of culture medium on the growth of MSC were analyzed. Mesenchymal stem calls were identified using the surface marker by flow cytometry. Osteoblast was identified by Von Kossa staining and alkaline phosphatase staining, adipocyte was identified by Oil Red O staining. Results When the other conditions were the same, the hespan isolation was better than Ficoll isolation; 5 × 10^6/cm^2 was the best planting density; the best first medium changing was on the seventh day at primary culture. Under optimized conditions, the MSC expressed adhesion molecules CD13, CD29and CD44 but not antigens of hematopoietic CD34, CD45, CD14 and not antigens of endothelia CD106. Exposure of these cells to osteogenic inductive agents resulted in an increase in expression of alkaline phosphatase and the appearance of hydroxyapatite nodules. Incubation with adipogenic inductive agents resulted in morphological change and staining with Oil Red O. Conclusion Mesenchymal stem cells exist in Cord blood, but slower to establish in culture. Cord blood may prove to be a new source of cells for cellular therapeutics. Keywords Umbilical cord blood; Mesenchymal stem cells; Culture