人脐带血造血干细胞的分离和体外扩增

目的 :通过对人新鲜脐带血造血干细胞的体外分离 ,探讨不同细胞生长因子的体外培养体系对CD34+ 细胞的扩增作用。方法 :用SCF、FL、GM CSF、IL 3、IL 6、G CSF、TPO组合成不同的实验组 ,对脐血中分离的CD34+ 细胞进行 2 1d的体外培养 ,扩增后经流式细胞仪分析 ,从而求得细胞培养较好的细胞因子组合。结果 :不同细胞因子组细胞培养孔CD34+ 细胞比例在培养 7d时已经开始上升 ,10d时继续上升 ,到 14d达高峰 ,继续培养 18d ,CD34+ 细胞比例逐渐下降 ,但仍高于培养前水平 ,培养 2 1d ,CD34+ 持续下降。总细胞数在第一周即扩增 35 .32倍 ,10～ 14d总细胞数扩增 331.7倍 ,2 1d总细胞数扩增 4 2 2 .2倍 ,SCF +IL 3+IL 6 +TPO +FL细胞因子组合对总细胞扩增最多 ,SCF +IL 3+IL 6 +GM CSF +G CSF对总细胞扩增略少 ,其次是SCF +TPO +FL细胞因子组合 ,最少为SCF +IL 3+IL 6 +GM CSF细胞因子组合 ,细胞涂片染色观察 ,体外培养 14d ,细胞胞体小 ,胞浆量少 ,胞核体积大 ,规则 ,为早期造血干细胞。结论 :体外HSC培养 ,细胞因子对细胞的增殖有明显的作用 ,但不同的细胞因子组合对细胞的扩增作用有明显的不同

Separating and Expansion of Hematopoietic Stem Cells of Human Umbilical Cord Blood in vitro

Objective To investigate the culture and expansion of the CD34 + hematopoietic stem cells(HSC) of human umbilical cord blood(UCB) in vitro using different cytokines. Methods Expanding CD34 + cells from the fresh umbilical cord blood for 21d in vitro with the use of different combination of FL,SCF,TPO,IL-3,IL-6,G-CSF and TPO. Getting one better combination by analysis of results from flowcytometry. Results The percentage of CD34 + cells which are in 24-well cells of different cytokine combinations begin to increase at the 7th day, and increase continuously at the 10th day, and reach the highest level at the 14th day. At the 18th day, the percentage of CD34 + decreased gradually, but still higher than the levels before cultivation. At the 21st day CD34 + decreased continuously. The total cell number increased by 35.32 times at the 1st week, and increased by 331.7 times at the 10～14th day, increased by 422.2 times at the 21st day. The combination of SCF+IL-3+IL-6+TPO+FL have greatest effect on the total cell number increasing, a little less is the combination of CF+IL-3+IL-6+GM-CSF+G-CSF. The second is SCF+TPO+FL combination, and the least one is the SCF+IL-3+IL-6+GM-CSF. It was found that the sizes of cells are small, the quantity of the cells pulp is small, the sizes of cell nuclear are large, and regular after the cell morphological detection observation and cultivating in vitro for 14 days. They were early-phrase hematopoietic stem cells. Conclusion The cultivation of the human cord blood HSC in vitro shows that different combinations of cytokines have different effects on cells expansion and the cytokines have an apparent promotion to the increase and expansion of mononuclear.