人脐血CD133^+细胞体外短期培养中生物学特性的变化

Studies on the Dynamics of Biological Characteristics of CD133^+ Cells from Human Umbilical Cord Blood during Short-term Culture

为了解脐血CD133+细胞的生物学特性及其在体外短期扩增培养中的变化 ,探讨其体外扩增的可行性 ,初步观察了脐血CD133+细胞的免疫表型、细胞周期、端粒酶活性以及粘附分子的表达等生物学特性及其在体外扩增中的动态变化并与CD34+细胞进行比较。结果显示 ,新鲜脐血CD133+和CD34+细胞的含量分别为 ( 1.0 5±0 73) %和 ( 1.4 0± 0 .5 6 ) % ,CD34+细胞中 79.6 2 %为CD133+CD34+细胞 ,而CD133+细胞中 97%以上为CD133+CD34+细胞。短期扩增培养结果显示 ,CD133+细胞组扩增第 10天 ,CD133+,CD133+CD34+和CD34+CD38-细胞以及第 6天的CFU mix ,HPP CFC和CD34+CD38-细胞的扩增倍数要高于CD34+细胞组 ( P <0 .0 5 ) ;扩增中 ,CD133+CD34+细胞的比例逐渐下降 ,而CD133-CD34+和CD133-CD34-细胞的比例则逐渐上升。新鲜脐血CD133+和CD34+细胞的端粒酶活性较低 ,但高于CD34-细胞。扩增 1周后 ,端粒酶活性明显上调 ,15天以后又逐渐下降 ;90 %以上脐血CD133+细胞表达CD11a ,CD4 9d和CD5 4 ,约 5 0 %表达CD6 2L。扩增早期 ,CD4 9d表达上调 ,CD11a表达无明显变化 ,而CD5 4和CD6 2L则有下调趋势 ,随着扩增时间的延长 ,各种粘附分子的表达均有不同程度的下调。在整个扩增过程中 ,大部分CD34+细胞仍然表达CD11a,CD4 9d和CD5 4?

This study was to investigate dynamics of biological properties of CD133 + cells from human umbilical cord blood (UCB) during short term culture containing the combination of hematopoietic growth factors and the feasibility of in vitro expansion of CD133 + cells.The biology activities including analysis of cell cycle, immunophenotype, telomerase activity, expression of adhesion molecules and expansion potential of CD133 + cells were monitored during ex vivo expansion, and compared with those of CD34 +cells. The results showed that the contents of CD133 + and CD34 + cells in fresh UCB were (1.05±0.73)% and (1.40±0.56)% respectively. About 79.62% of CD34 + cells expressed CD133, and more than 97% of CD133 +cells were CD133 +CD34 +, markedly higher than that in CD34 + fraction (P<0.01). No significant differences were observed in content of cells expressing CD38, CD13, CD14, CD61 and glycophorin A between the two fractions. Expansion of CD133 +, CD133 +CD34 + and CD34 +CD38 - cells at 10 days and those of CFU mix, HPP CFC and CD34 +CD38 - cells at 6 days from CD133 +cells group were significantly higher than those from the CD34 + cell group (P<0.05). Analysis of immunophenotype showed that CD133 +CD34 + cells declined gradually while CD133 -CD34 + and CD133 -CD34 - cells increased during ex vivo expansion; basal telomerase activities of fresh UCB CD133 + and CD34 + cells were low but significantly exceeded that of CD34 - fraction (P<0.05). At first week of expansion, telomerase activity was significantly upregulated, after two weeks, telomerase activity remarkably declined, and decreased to baseline or below the limits of detection in day 20. More than 90% of CD133 +cells expressed CD49d and CD11a, and, more than 85% of the cells expressed CD54, about 50% of cells expressed CD62L. At the early stage of expansion, expression of CD49d was upregulated, expression of CD11a remaining no change, while as expression of CD54 and CD62L was downregulated. Expression of all adhesion molecules was decreased gradually with extend of culture. But expression of these adhesion molecules on CD34 + subsets were not affected significantly during expansion. It is concluded that CD133 + population may be a more primitive hematopoietic stem/progenitor cells (HSPC) than CD34 + cells, CD133 +cells have great expansion potential for ex vivo expansion and is a suitable target cell for ex vivo expansion of HSPC. Downregulation of adhesion molecules and telomerase activity may be one of the reasons for delayed engraftment of expanded products.