Xia Ting, Fang Jian-Pei, Wu Yan-Feng, Xu Hong-Gui, Wei Qing
Department of Pediatrics, Guangzhou Maternal and Neonatal Hospital, Guangzhou 510180, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2006 Aug;14(4):745-8.
This study was aimed to investigate the effect of some culture system composed of various cytokine combinations (TPO, SCF, FL, IL-1, IL-3, IL-6) on ex vivo expansion of megakaryocytic progenitors induced from CD34+ cells of peripheral blood and to seek a optimal cytokine combination and culture time. Mononuclear cells were isolated from mobilized peripheral blood (MPB) by density gradient centrifugation over Ficoll. CD34+ cells were purified by using an immunomagnetic bead separation system. The selected CD34+ cells were seeded in Iscove's modified Kulbecco's medium (IMDM) supplemented with fetal calf serum (FCS) and various kinds of cytokines. After 15 days of culture, the content of CD41+ cells in culture system were determined by flow cytometry, and the number of megakaryocyte colony-forming unit (CFU-MK) was measured simultaneously. The results showed that after definited days of culture, the cytokine combination TPO/FL/IL-6/IL-3 was the most suitable for MPB to obtain high number of MK, and better than any other three groups (P < 0.05). The increase multiple of CD41+ cells was 93.97 +/- 17.27 on day 5 and 131.23 +/- 18.26 on day 10. On day 15, the proportion and the increase multiple of CD41+ cells decreased obviously. The expansion multiples of CFU-MK were 93.33 +/- 10.02 on day 5 and 120.67 +/- 13.01 on day 10, higher than any other groups. It is concluded that TPO/FL/IL-6/IL-3 combination was the best optimal for expansion ex vivo of megakaryocytic progenitors from MPB, and its suitable duration of culture was 10 days; a culture system for expansion ex vivo of megakaryocytic progenitors have been established in this study.
本研究旨在探讨由多种细胞因子组合（血小板生成素、干细胞因子、Flt3配体、白细胞介素-1、白细胞介素-3、白细胞介素-6）组成的培养体系对外周血CD34+细胞诱导的巨核细胞祖细胞体外扩增的影响，并寻找最佳的细胞因子组合和培养时间。通过Ficoll密度梯度离心从动员的外周血（MPB）中分离单个核细胞。使用免疫磁珠分离系统纯化CD34+细胞。将筛选出的CD34+细胞接种于添加胎牛血清（FCS）和各种细胞因子的Iscove改良杜氏培养基（IMDM）中。培养15天后，通过流式细胞术检测培养体系中CD41+细胞的含量，并同时测定巨核细胞集落形成单位（CFU-MK）的数量。结果显示，培养一定天数后，细胞因子组合血小板生成素/Flt3配体/白细胞介素-6/白细胞介素-3最适合MPB获得大量巨核细胞，且优于其他三组（P<0.05）。第5天CD41+细胞的增加倍数为93.97±17.27，第10天为131.23±18.26。第15天，CD41+细胞的比例和增加倍数明显下降。CFU-MK的扩增倍数第5天为93.33±10.02，第10天为120.67±13.01，高于其他任何组。结论是血小板生成素/Flt3配体/白细胞介素-6/白细胞介素-3组合是MPB来源的巨核细胞祖细胞体外扩增的最佳组合，其适宜培养时间为10天；本研究建立了一种巨核细胞祖细胞体外扩增的培养体系。
